Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins

Covalently linked cell wall proteins (CWPs) of the dimorphic fungus Candida albicans are implicated in virulence. We have carried out a comprehensive proteomic analysis of the covalently linked CWPs in exponential-phase yeast cells. Proteins were liberated from sodium dodecyl sulfate (SDS)-extracted cell walls and analyzed using immunological and advanced protein sequencing (liquid chromatography-tandem mass spectrometry [LC/MS/MS]) methods. HF-pyridine and NaOH were used to chemically release glycosylphosphatidylinositol-dependent proteins (GPI proteins) and mild alkali-sensitive proteins, respectively. In addition, to release both classes of CWPs simultaneously, cell walls were digested enzymatically with a recombinant beta-1,3-glucanase. Using LC/MS/MS, we identified 14 proteins, of which only 1 protein, Cht2p, has been previously identified in cell wall extracts by using protein sequencing methods. The 14 identified CWPs include 12 GPI proteins and 2 mild alkali-sensitive proteins. Nonsecretory proteins were absent in our cell wall preparations. The proteins identified included several functional categories: (i) five CWPs are predicted carbohydrate-active enzymes (Cht2p, Crh11p, Pga4p, Phr1p, and Scw1p); (ii) Als1p and Als4p are believed to be adhesion proteins. In addition, Pga24p shows similarity to the flocculins of baker's yeast. (iii) Sod4p/Pga2p is a putative superoxide dismutase and is possibly involved in counteracting host defense reactions. The precise roles of the other CWPs (Ecm33.3p, Pir1p, Pga29p, Rbt5p, and Ssr1p) are unknown. These results indicate that a substantial number of the covalently linked CWPs of C. albicans are actively involved in cell wall remodeling and expansion and in host-pathogen interactions.     

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants

In Candida albicans wild-type cells, the beta1, 6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via beta1,6-glucan to beta1,3-glucan. The remaining GPI-CWPs are linked through beta1,6-glucan to chitin. The beta1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to beta1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Delta and pmt1Delta mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through beta1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a beta1, 6-glucan-deficient hemizygous kre6Delta mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.     

Comprehensive proteomic analysis of Saccharomyces cerevisiae cell walls: identification of proteins covalently attached via glycosylphosphatidylinositol remnants or mild alkali-sensitive linkages

The cell wall of yeast contains proteins that are covalently bound to the glycan network. These cell wall proteins (CWPs) mediate cell-cell interactions and may be involved in cell wall biosynthesis. Using tandem mass spectrometry, we have identified 19 covalently bound CWPs of Saccharomyces cerevisiae. Twelve of them are shown for the first time to be covalently incorporated into the cell wall. The identified proteins include 12 predicted glycosylphosphatidylinositol-modified CWPs, all four members of the Pir protein family, and three additional proteins (Scw4p, Scw10p, and Tos1p) that are, like Pir proteins, connected to the cell wall glycan network via an alkali-sensitive linkage. However, Scw4p, Scw10p, and Tos1p do not contain internal repeat sequences shown to be essential for Pir protein incorporation and may represent a separate class of CWPs. Strikingly, seven of the identified proteins (Gas1p, Gas3p, Gas5p, Crh1p, Utr2p, Scw4p, and Scw10p) are classified as glycoside hydrolases. Phenotypic analysis of deletion mutants lacking the corresponding CWP-encoding genes indicated that most of them have altered cell wall properties, which reinforces the importance of the identified proteins for proper cell wall formation. In particular, gas1Delta and ecm33Delta were highly sensitive to Calcofluor White and high temperature, whereas gas1Delta, scw4Delta, and tos1Delta were highly resistant to incubation with beta-1,3-glucanase. The CWP identification method developed here relies on directly generating tryptic peptides from isolated cell walls and is independent of the nature of the covalent linkages between CWPs and cell wall glycans. Therefore, it will probably be equally effective in many other fungi.     

Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae

The cell wall of yeast consists of an internal skeletal layer and an external layer of glycoproteins covalently linked to the stress-bearing polysaccharides. The cell wall protein (CWP) population consists of over 20 different proteins, and may vary in composition. We present two complementary methods for quantifying CWPs, based on isobaric tagging and tandem MS: (1) absolute quantitation of individual CWPs, allowing estimation of surface densities; and (2) relative quantitation of CWPs, allowing monitoring of the dynamics of the CWP population. For absolute quantitation, we selected a representative group of five proteins (Cwp1p, Crh1p, Scw4p, Gas1p, and Ecm33p), which had 67 x 10(3), 44 x 10(3), 38 x 10(3), 11 x 10(3) and 6.5 x 10(3) of wall-bound copies per cell, respectively. As Cwp1p is predominantly incorporated in the birth scar, this corresponds to a protein density of c. 22 x 10(3) copies microm(-2). For relative quantitation, we compared wild-type cells to gas1Delta cells, in which the cell wall integrity pathway is constitutively activated. The levels of Crh1p, Crh2p, Ecm33p, Gas5p, Pst1p and Pir3p increased about three- to fivefold, whereas the level of Scw4p was significantly decreased. We propose that our methods are widely applicable to other fungi.     

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

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.     

Proteolytic cleavage of covalently linked cell wall proteins by Candida albicans Sap9 and Sap10

The cell wall of the human-pathogenic fungus Candida albicans is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surface-associated proteases which function in C. albicans cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues. However, both proteases also cleaved at nonbasic sites, and not all tested peptides with dibasic residues were processed. By digesting isolated cell walls with Sap9 or Sap10, we identified the covalently linked cell wall proteins (CWPs) Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 as in vitro protease substrates. Proteolytic cleavage of the chitinase Cht2 and the glucan-cross-linking protein Pir1 by Sap9 was verified using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the SAP9 and SAP10 genes resulted in a reduction of cell-associated chitinase activity similar to that upon deletion of CHT2, suggesting a direct influence of Sap9 and Sap10 on Cht2 function. In contrast, cell surface changes elicited by SAP9 and SAP10 deletion had no major impact on the phagocytosis and killing of C. albicans by human macrophages. We propose that Sap9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins.     

Use of green fluorescent protein fusions to analyse the N- and C-terminal signal peptides of GPI-anchored cell wall proteins in Candida albicans

Glycophosphatidylinositol (GPI)-anchored proteins account for 26-35% of the Candida albicans cell wall. To understand the signals that regulate these proteins' cell surface localization, green fluorescent protein (GFP) was fused to the N- and C-termini of the C. albicans cell wall proteins (CWPs) Hwp1p, Als3p and Rbt5p. C. albicans expressing all three fusion proteins were fluorescent at the cell surface. GFP was released from membrane fractions by PI-PLC and from cell walls by beta-glucanase, which implied that GFP was GPI-anchored to the plasma membrane and then covalently attached to cell wall glucans. Twenty and 25 amino acids, respectively, from the N- and C-termini of Hwp1p were sufficient to target GFP to the cell surface. C-terminal substitutions that are permitted by the omega rules (G613D, G613N, G613S, G613A, G615S) did not interfere with GFP localization, whereas some non-permitted substitutions (G613E, G613Q, G613R, G613T and G615Q) caused GFP to accumulate in intracellular ER-like structures and others (G615C, G613N/G615C and G613D/G615C) did not. These results imply that (i) GFP fusions can be used to analyse the N- and C-terminal signal peptides of GPI-anchored CWPs, (ii) the omega amino acid in Hwp1p is G613, and (iii) C can function at the omega+2 position in C. albicans GPI-anchored proteins.     

Cell wall proteome in the maize primary root elongation zone. I. Extraction and identification of water-soluble and lightly ionically bound proteins

Cell wall proteins (CWPs) play important roles in various processes, including cell elongation. However, relatively little is known about the composition of CWPs in growing regions. We are using a proteomics approach to gain a comprehensive understanding of the identity of CWPs in the maize (Zea mays) primary root elongation zone. As the first step, we examined the effectiveness of a vacuum infiltration-centrifugation technique for extracting water-soluble and loosely ionically bound (fraction 1) CWPs from the root elongation zone. The purity of the CWP extract was evaluated by comparing with total soluble proteins extracted from homogenized tissue. Several lines of evidence indicated that the vacuum infiltration-centrifugation technique effectively enriched for CWPs. Protein identification revealed that 84% of the CWPs were different from the total soluble proteins. About 40% of the fraction 1 CWPs had traditional signal peptides and 33% were predicted to be nonclassical secretory proteins, whereas only 3% and 11%, respectively, of the total soluble proteins were in these categories. Many of the CWPs have previously been shown to be involved in cell wall metabolism and cell elongation. In addition, maize has type II cell walls, and several of the CWPs identified in this study have not been identified in previous cell wall proteomics studies that have focused only on type I walls. These proteins include endo-1,3;1,4-beta-D-glucanase and alpha-L-arabinofuranosidase, which act on the major polysaccharides only or mainly present in type II cell walls.     

Cell wall proteins of the ectomycorrhizal basidiomycete Pisolithus tinctorius: identification, function, and expression in symbiosis.

Specific cell-cell and cell-substrate interactions direct the growth of ectomycorrhizal fungi to their host root targets. These elaborate mechanisms lead to the differentiation of distinct multihyphal structures, the mantle, and the Hartig net. In the ectomycorrhizal basidiomycete Pisolithus tinctorius, the use of two-dimensional gel electrophoresis, immunocytochemical microscopy, and RNA blot analysis has demonstrated the differential expression of cell wall proteins (CWPs), such as hydrophobins, adhesins, and mannoproteins, during symbiotic interaction. In other fungi, these CWPs have been suggested to play a role in hyphae aggregation, intracellular signaling cascades, and cytoskeletal changes. The recent cloning of the genes for several of these CWPs in P. tinctorius allows us to address their function in symbiosis. This review summarizes our knowledge of CWPs in P. tinctorius and considers parallels with other biotrophic fungi as a possible framework for future work.     

Characterization of agglutinin-like sequence genes from non-albicans Candida and phylogenetic analysis of the ALS family

The ALS (agglutinin-like sequence) gene family of Candida albicans encodes cell-surface glycoproteins implicated in adhesion of the organism to host surfaces. Southern blot analysis with ALS-specific probes suggested the presence of ALS gene families in C. dubliniensis and C. tropicalis; three partial ALS genes were isolated from each organism. Northern blot analysis demonstrated that mechanisms governing expression of ALS genes in C. albicans and C. dubliniensis are different. Western blots with an anti-Als serum showed that cross-reactive proteins are linked by beta 1,6-glucan in the cell wall of each non-albicans Candida, suggesting similar cell wall architecture and conserved processing of Als proteins in these organisms. Although an ALS family is present in each organism, phylogenetic analysis of the C. albicans, C. dubliniensis, and C. tropicalis ALS genes indicated that, within each species, sequence diversification is extensive and unique ALS sequences have arisen. Phylogenetic analysis of the ALS and SAP (secreted aspartyl proteinase) families show that the ALS family is younger than the SAP family. ALS genes in C. albicans, C. dubliniensis, and C. tropicalis tend to be located on chromosomes that also encode genes from the SAP family, yet the two families have unexpectedly different evolutionary histories. Homologous recombination between the tandem repeat sequences present in ALS genes could explain the different histories for co-localized genes in a predominantly clonal organism like C. albicans.     

Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence

During infection of their hosts, Gram-positive bacteria express surface proteins that serve multiple biological functions. Surface proteins harbouring a C-terminal sorting signal with an LPXTG motif are covalently linked to the cell wall peptidoglycan by a transamidase named sortase. Two genes encoding putative sortases, termed srtA and srtB, were identified in the genome of the intracellular pathogenic bacterium Listeria monocytogenes. Inactivation of srtA abolishes anchoring of the invasion protein InlA to the bacterial surface. It also prevents the proper sorting of several other peptidoglycan-associated LPXTG proteins. Three were identified by a mass spectrometry approach. The DeltasrtA mutant strain is defective in entering epithelial cells, similar to a DeltainlA mutant. In contrast to a DeltainlA mutant, the DeltasrtA mutant is impaired for colonization of the liver and spleen after oral inoculation in mice. Thus, L. monocytogenes srtA is required for the cell wall anchoring of InlA and, presumably, for the anchoring of other LPXTG-containing proteins that are involved in listerial infections.     

Features and functions of covalently linked proteins in fungal cell walls

The cell walls of many ascomycetous yeasts consist of an internal network of stress-bearing polysaccharides, which serve as a scaffold for a dense external layer of glycoproteins. GPI-modified proteins are the most abundant cell wall proteins and often display a common organization. Their C-terminus can link them covalently to the polysaccharide network, they possess an internal serine- and threonine-rich spacer domain, and the N-terminal region contains a functional domain. Other proteins bind to the polysaccharide network through a mild-alkali-sensitive linkage. Many cell wall proteins are carbohydrate/glycan-modifying enzymes; adhesion proteins are prominent; proteins involved in iron uptake are present, and also specialized proteins that probably help the fungus to survive in its natural environment. The protein composition of the cell wall depends on environmental conditions and developmental stage. We present evidence that the cell wall of mycelial species of the Ascomycotina is similarly organized and contains glycoproteins with comparable functions.     

Cell wall proteomics contributes to explore the functional proteins of Brachypodium distachyon grains

The plant cell wall is the first barrier in response to external stimuli and cell wall proteins (CWPs) can play an important role in the modulation of plant growth and development. In the past 10 years, the plant cell wall proteomics has increasingly become a very active research filed, which provides a broader understanding of CWPs for people. The cell wall proteome of Arabidopsis, rice, and other model plants has begun to take shape, and proteomic technology has become an effective way to identify the candidate functional CWPs in large scale. The challenging work of Francin‐Allami et al. (Proteomics 2015, 15, 2296–2306) is a vital step toward building the most extensive cell wall proteome of a monocot species. They identified 299 cell wall proteins in Brachypodium distachyon grains, and also compared the grain cell wall proteome with those of B. distachyon culms and leaves, which provides a new perspective for further explaining the plant cell wall structures and remodeling mechanism.     

Cell wall proteomics of the green alga Haematococcus pluvialis (Chlorophyceae)

The green microalga Haematococcus pluvialis can synthesize and accumulate large amounts of the ketocarotenoid astaxanthin, and undergo profound changes in cell wall composition and architecture during the cell cycle and in response to environmental stresses. In this study, cell wall proteins (CWPs) of H. pluvialis were systematically analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with peptide mass fingerprinting (PMF) and sequence-database analysis. In total, 163 protein bands were analyzed, which resulted in positive identification of 81 protein orthologues. The highly complex and dynamic composition of CWPs is manifested by the fact that the majority of identified CWPs are differentially expressed at specific stages of the cell cycle along with a number of common wall-associated 'housekeeping' proteins. The detection of cellulose synthase orthologue in the vegetative cells suggested that the biosynthesis of cellulose occurred during primary wall formation, in contrast to earlier observations that cellulose was exclusively present in the secondary wall of the organism. A transient accumulation of a putative cytokinin oxidase at the early stage of encystment pointed to a possible role in cytokinin degradation while facilitating secondary wall formation and/or assisting in cell expansion. This work represents the first attempt to use a proteomic approach to investigate CWPs of microalgae. The reference protein map constructed and the specific protein markers obtained from this study provide a framework for future characterization of the expression and physiological functions of the proteins involved in the biogenesis and modifications in the cell wall of Haematococcus and related organisms.     

Cell Wall Proteome of Sugarcane Young and Mature Leaves and Stems

By characterizing the cell wall proteomes of different sugarcane organs (leaves and stems) at two developmental stages (young vs mature/apical vs basal), it is possible to address unique characteristics in each of them. Four‐month‐old leaves show a higher proportion of oxido‐reductases and proteins related to lipid metabolism (LM), besides a lower proportion of proteins acting on polysaccharides, in comparison to 4‐month‐old internodes. It is possible to note that sugarcane leaves and young stems have the highest LM rate than all species, which is assumed to be linked to cuticle formation. The data generated enrich the number of cell wall proteins (CWPs) identified in sugarcane, reaching 277. To our knowledge, sugarcane has now the second higher coverage of monocot CWP in plants.     

Antibody response to Candida albicans cell wall antigens

The cell wall of Candida albicans is not only the structure where many essential biological functions reside but is also a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both carbohydrate and protein moieties are able to trigger immune responses. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to profoundly influence the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins to host ligands. In this review we examine various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo. Some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidiasis, particularly the disseminated form. In addition, recent studies have focused on the potential of antibodies against the cell wall protein determinants in protecting the host against infection. Hence, a better understanding of the humoral response triggered by the cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the serodiagnosis of disseminated candidiasis, and (ii) novel prophylactic (vaccination) and therapeutic strategies to control this type of infections.     

Immunoproteomic analysis of the protective response obtained from vaccination with Candida albicans ecm33 cell wall mutant in mice

Systemic candidiasis remains a major cause of disease and death, particularly among immunocompromised patients. The cell wall of Candida albicans defines the interface between host and pathogen and surface proteins are major elicitors of host immune responses during candidiasis. The C. albicans ecm33 mutant (RML2U) presents an altered cell wall, which entails an increase in the outermost protein layer. Vaccination of BALB/c mice with RML2U mutant protected them from a subsequent lethal infection with virulent strain SC5314 in a systemic candidiasis model. Using immunoproteomics (2-DE followed by Immunoblotting) we detected 29 immunoreactive proteins specifically recognized by antibodies from vaccinated mice sera, six of which are described as immunogenic for the first time (Gnd1p, Cit1p, Rpl10Ep, Yst1p, Cys4p, Efb1p). Furthermore, identification of wild type and mutant cell surface proteome (surfome), confirmed us that the mutant surfome presented a larger number of proteins than the wild type. Interestingly, proteins exclusively identified in the mutant surfome (Met6p, Eft2p, Tkl1p, Rpl10Ep, Atp1p, Atp2p) were also detected as immunogenic, supporting the idea that their surface location enhances their immunoprotective capacity.