Aida Pitarch

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.

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

In an effort to bring novel diagnostic and prognostic biomarkers or even potential targets for vaccine design for systemic candidiasis (SC) into the open, a systematic proteomic approach coupled with bioinformatic analysis was used to decode the serological response to Candida wall immunome in SC patients. Serum levels of IgG antibodies against Candida wall-associated proteins (proteins secreted from protoplasts in active wall regeneration, separated by two-dimensional gel electrophoresis, and identified by mass spectrometry) were measured in 45 SC patients, 57 non-SC patients, and 61 healthy subjects by Western blotting. Two-way hierarchical clustering and principal component analysis of their serum anti-Candida wall antibody expression patterns discriminated SC patients from controls and highlighted the heterogeneity of their expression profiles. Multivariate logistic regression models demonstrated that high levels of antibodies against glucan 1,3-β-glucosidase (Bgl2p) and the anti-wall phosphoglycerate kinase antibody seropositivity were the only independent predictors of SC. Receiver operating characteristic curve analysis revealed no difference between their combined evaluation and measurement of anti-Bgl2p antibodies alone. In a logistic regression model adjusted for known prognostic factors for mortality, SC patients with high anti-Bgl2p antibody levels or a positive anti-wall enolase antibody status, which correlated with each other, had a reduced 2-month risk of death. After controlling for each other, only the seropositivity for anti-wall enolase antibodies was an independent predictor of a lower risk of fatality, supporting that these mediated the protective effect. No association between serum anti-cytoplasmic enolase antibody levels and outcomes was established, suggesting a specific mechanism of enolase processing during wall biogenesis. We conclude that serum anti-Bgl2p antibodies are a novel accurate diagnostic biomarker for SC and that, at high levels, they may provide protection by modulating the anti-wall enolase antibody response. Furthermore serum anti-wall enolase antibodies are a new prognostic indicator for SC and confer protection against it. Bgl2p and wall-associated enolase could be valuable candidates for future vaccine development.

Cross-species identification of novel Candida albicans immunogenic proteins by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

We have previously reported the usefulness of two‐dimensional gel electrophoresis followed by Western blotting with sera from patients with systemic candidiasis in the detection of the major Candida albicans antigens (Pitarch et al., Electrophoresis 1999, 20, 1001—1010). The identification of these antigens would be useful for the characterization of good markers for the disease, and for the development of efficient diagnostic strategies. In this work we have used nanoelectrospray tandem mass spectrometry to obtain amino acid sequence information from the immunogenic proteins previously detected. We report here the cross‐species identification of these antigens by matching of tandem mass spectrometry data to Saccharomyces cerevisiae proteins. Using this approach, we unambiguously identified the four C. albicans immunogenic proteins analyzed, namely aconitase, pyruvate kinase, phosphoglycerate mutase and methionine synthase. Furthermore, we report for the first time that aconitase, methionine synthase and phosphoglycerate mutase have antigenic properties in C. albicans.

Cell wall fractionation for yeast and fungal proteomics.

The cell wall is an external envelope shared by yeasts and filamentous fungi that defines the interface between the microorganism and its environment. It is an extremely complex structure consisting of an elastic framework of microfibrillar polysaccharides (glucans and chitin) that surrounds the plasma membrane and to which a wide array of different proteins, often heavily glycosylated, are anchored in various ways. Intriguingly, these cell wall proteins (CWPs) play a key role in morphogenesis, adhesion, pathogenicity, antigenicity, and as a promising target for antifungal drug design. However, the CWPs are difficult to analyze because of their low abundance, low solubility, hydrophobic nature, extensive glycosylation, covalent attachment to the wall polysaccharide skeleton, and high heterogeneity. We describe a typical procedure of cell wall fractionation to isolate and solubilize different CWP species from yeasts and filamentous fungi according to the type of linkages that they establish with other wall components and under suitable conditions for following reproducible proteomic analyses. CWPs retained noncovalently or by disulfide bonds are first extracted from isolated yeast or fungal cell walls by detergents and reducing agents. Subsequently, CWPs covalently linked to or closely entrapped within the internal glucan-chitin network are sequentially released either by mild alkali conditions or by enzymatic treatments first with glucanases and then with chitinases. This strategy is a powerful tool not only for obtaining an overview of the sophisticated cell wall proteome of yeasts and filamentous fungi, but also for characterizing mechanisms of incorporation, assembly and retention of CWPs into this intricate cellular compartment and their interactions with structural wall polysaccharides.

Prediction of the Clinical Outcome in Invasive Candidiasis Patients Based on Molecular Fingerprints of Five Anti-Candida Antibodies in Serum

Better prognostic predictors for invasive candidiasis (IC) are needed to tailor and individualize therapeutic decision-making and minimize its high morbidity and mortality. We investigated whether molecular profiling of IgG-antibody response to the whole soluble Candida proteome could reveal a prognostic signature that may serve to devise a clinical-outcome prediction model for IC and contribute to known IC prognostic factors. By serological proteome analysis and data-mining procedures, serum 31-IgG antibody-reactivity patterns were examined in 45 IC patients randomly split into training and test sets. Within the training cohort, unsupervised two-way hierarchical clustering and principal-component analyses segregated IC patients into two antibody-reactivity subgroups with distinct prognoses that were unbiased by traditional IC prognostic factors and other patients-related variables. Supervised discriminant analysis with leave-one-out cross-validation identified a five-IgG antibody-reactivity signature as the most simplified and accurate IC clinical-outcome predictor, from which an IC prognosis score (ICPS) was derived. Its robustness was confirmed in the test set. Multivariate logistic-regression and receiver-operating-characteristic curve analyses demonstrated that the ICPS was able to accurately discriminate IC patients at high risk for death from those at low risk and outperformed conventional IC prognostic factors. Further validation of the five-IgG antibody-reactivity signature on a multiplexed immunoassay supported the serological proteome analysis results. The five IgG antibodies incorporated in the ICPS made biologic sense and were associated either with good-prognosis and protective patterns (those to Met6p, Hsp90p, and Pgk1p, putative Candida virulence factors and antiapoptotic mediators) or with poor-prognosis and risk patterns (those to Ssb1p and Gap1p/Tdh3p, potential Candida proapoptotic mediators). We conclude that the ICPS, with additional refinement in future larger prospective cohorts, could be applicable to reliably predict patient clinical-outcome for individualized therapy of IC. Our data further provide insights into molecular mechanisms that may influence clinical outcome in IC and uncover potential targets for vaccine design and immunotherapy against IC.

Analysis of the Candida albicans proteome: I. Strategies and applications

The alarming incidence of invasive candidiasis, predominantly among the recent expanding immunocompromised population, the appearance of antifungal-drug resistance, and the lack of specific diagnostic tests for it have demanded more impactful research into Candida albicans pathogenicity. Proteomic approaches can provide accurate clues about its biological complexity. Indeed, initial C. albicans proteome analyses have focused on the understanding of dimorphism, host responses, the cell wall, virulence factors and drug resistance, among others. This review aims to briefly outline the technology available for proteomics-based studies, surveying the main proteomic approaches applied to C. albicans research. Prefractionation techniques, two-dimensional gel electrophoresis and mass spectrometry continue to be the backbone of proteomic projects. Emerging strategies for protein separation, quantification and identification may, however, challenge the pivotal position of 2D-PAGE. Regardless of this, since we are now approaching the completion and annotation of C. albicans genome sequencing, systematic characterization of the proteome of this fungal pathogen, although still in its early stages, heralds an exciting expansion of our knowledge in years to come.

Quantitative proteome and acidic subproteome profiling of Candida albicans yeast-to-hypha transition.

Candida albicans yeast-to-hypha morphological transition is involved in the virulence strategy of this opportunistic fungal pathogen. Changes in relative abundance of the Candida proteome related to this process were analyzed using different two-dimensional differential in-gel electrophoresis (2D-DIGE)-based approaches. First, a comparative analysis of yeast and hyphal cytoplasmic proteins allowed the detection of 106 protein spots with significant variation in abundance. Sixty-one of them, corresponding to 46 proteins, were identified. As most of the differentially abundant proteins had an acidic isoelectric point, a large-scale prefractionation approach to analyze the acidic C. albicans subproteome was carried out. Ninety acidic C. albicans proteins were identified by either gel-based or nongel-based approaches. Additionally, different workflows combining preparative isoelectric focusing, Cy labeling, and narrow pH gradient 2-DE gels were tested to analyze the differences in relative protein abundance between yeast and hyphal acidic subproteomes. It was possible to identify 21 differentially abundant acidic proteins; 10 of them were not identified in the previous 2D-DIGE gels. Functional and network interaction analyses of the 56 differentially abundant proteins identified by both approaches rendered an integrated view of metabolic and cellular process reorganization during the yeast-to-hypha transition. With these results, we propose a model of metabolic reorganization.

Proteomics to Study Candida albicans Biology and Pathogenicity

Candida albicans is an opportunistic pathogenic fungus capable of causing infections in an expanding population of immunosuppressed patients. The implementation of proteomics in the post-genomic era of this organism can provide vital information about its biological complexity and pathogenic traits. C. albicans proteomic analyses to date have focused on the understanding of the cell wall, virulence, dimorphism, antifungal drug effects and resistance, and serological response, among others. This exciting and rapid growing discipline should become an indispensable tool in C. albicans research, particularly to address problems that cannot be solved by genomic studies. Furthermore, in the near future it is expected that results from proteomic experiments will lead to novel techniques for the management of candidiasis.

Proteomic Profiling of Serologic Response to Candida albicans During Host-Commensal and Host-Pathogen Interactions

Candida albicans is a commensal inhabitant of the normal human microflora that can become pathogenic and invade almost all body sites and organs in response to both host-mediated and fungus-mediated mechanisms. Serologic responses to C. albicans that underlie its dichotomist relationship with the host (host-commensal and host-pathogen interactions) display a high degree of heterogeneity, resulting in distinct serum anti-Candida antibody signatures (molecular fingerprints of anti-Candida antibodies in serum) that can be used to discriminate commensal colonization from invasive disease. We describe the typical proteomic strategy to globally and integratively profile these host antibody responses and determine serum antibody signatures. This approach is based on the combination of classic immunoproteomics or serologic proteome analysis (two-dimensional electrophoresis followed by quantitative Western blotting and mass spectrometry) with data mining procedures. This global proteomic stratagem is a useful tool not only for obtaining an overview of different anti-Candida antibodies that are being elicited during the host-fungus interaction and, consequently, of the complex C. albicans immunome (the subset of the C. albicans proteome targeted by the immune system), but also for evaluating how this pathogen organism interacts with its host to trigger infection. In contrast with genomics and transcriptomics, this proteomic technology has the potential to detect antigenicity associated with posttranslational modification, subcellular localization, and other functional aspects that can be relevant in the host immune response. Furthermore, this strategy to define molecular fingerprints of serum anti-Candida antibodies may hopefully bring to light potential candidates for diagnosis, prognosis, risk stratification, clinical follow-up, therapeutic monitoring, and/or immunotherapy of candidiasis, especially of its life-threatening systemic forms.

Serological proteome analysis to identify systemic candidiasis patients in the intensive care unit : Analytical, diagnostic and prognostic validation of anti-Candida enolase antibodies on quantitative clinical platforms

Systemic candidiasis (SC) is associated with high morbidity and mortality, because it generally affects patients with severe underlying diseases and its diagnosis is difficult and often delayed, resulting in delayed therapy. We used serological proteome analysis to screen serum anti‐Candida IgG antibody‐reactivity profiles in 24 patients under intensive care, 12 of which had confirmed SC (fungal cultures), and in 12 healthy subjects. A total of 15 immunogenic proteins from Candida albicans protoplast lysates were differentially immunorecognized by serum IgG antibodies from SC patients compared to controls. Two‐way hierarchical clustering and principal‐component analyses of these antibody‐reactivity patterns accurately differentiated SC patients from controls. Anti‐Eno1p IgG antibodies were found to be present at high abundance in SC patients and be an important molecular fingerprint in serum for SC diagnosis. Differential anti‐Eno1p IgG antibody reactivity was further validated by a tag capture ELISA and a Western blot assay in 45 SC patients and 118 non‐SC subjects. Both quantitative assays provided comparable analytical, diagnostic and prognostic performances, and verified initial proteomic‐profiling results. If confirmed in prospective cohort studies, these anti‐Eno1p IgG antibodies might be useful for SC diagnosis. However, these, at least as measured by these clinical platforms, appear to have limited prognostic value in SC patients.