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ABSTRACT: We used a brief trypsin treatment followed by peptide separation and identification using nano-LC followed by off-line MS/MS to identify the surface proteins on live Candida albicans organisms growing in biofilms and planktonic yeast cells and hyphae. One hundred thirty-one proteins were present in at least two of the three replicates of one condition and distributed in various combinations of the three growth conditions. Both previously reported and new surface proteins were identified and these were distributed between covalently attached proteins and noncovalently attached proteins of the cell wall.
Proteomics 06/2012; 12(14):2331-9. · 4.43 Impact Factor
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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.
Journal of proteomics 02/2012; 75(15):4734-46. · 5.07 Impact Factor
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ABSTRACT: The following report provides an overview of the discussions and outcome of the EuPA General Council meeting that took place in Estoril 20-21 October 2010. During the annual meeting future policy and action plans in a variety of areas are decided. Several important points were decided upon during this meeting including the expansion of the EuPA Executive Committee by introducing a new EuPA committee - EuPA Developments - that will initially spearhead activities in standardisation, imaging ms and biobanking. The EuPA General Council also invited Russia as its 17th member. More details about these and additional activities are presented in the article.
Journal of proteomics 06/2011; 75(1):18-22. · 5.07 Impact Factor
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ABSTRACT: 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.
Journal of Proteome Research 02/2011; 10(2):502-17. · 5.11 Impact Factor
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ABSTRACT: 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.
Molecular & Cellular Proteomics 01/2011; 10(1):M110.004010. · 7.40 Impact Factor
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Enfermedades Infecciosas y Microbiología Clínica 10/2010; 28(8):489-91. · 1.49 Impact Factor
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ABSTRACT: Aspergillus nidulans early endosomes display characteristic long-distance bidirectional motility. Simultaneous dual-channel acquisition showed that the two Rab5 paralogues RabB and RabA colocalize in these early endosomes and also in larger, immotile mature endosomes. However, RabB-GTP is the sole recruiter to endosomes of Vps34 PI3K (phosphatidylinositol-3-kinase) and the phosphatidylinositol-3-phosphate [PI(3)P] effector AnVps19 and rabB Delta, leading to thermosensitivity prevents multivesicular body sorting of endocytic cargo. Thus, RabB is the sole mediator of degradative endosomal identity. Importantly, rabB Delta, unlike rabA Delta, prevents early endosome movement. As affinity experiments and pulldowns showed that RabB-GTP recruits AnVps45, RabB coordinates PI(3)P-dependent endosome-to-vacuole traffic with incoming traffic from the Golgi and with long-distance endosomal motility. However, the finding that Anvps45 Delta, unlike rabB Delta, severely impairs growth indicates that AnVps45 plays RabB-independent functions. Affinity chromatography showed that the CORVET complex is a RabB and, to a lesser extent, a RabA effector, in agreement with GST pulldown assays of AnVps8. rabB Delta leads to smaller vacuoles, suggesting that it impairs homotypic vacuolar fusion, which would agree with the sequential maturation of endosomal CORVET into HOPS proposed for Saccharomyces cerevisiae. rabB Delta and rabA Delta mutations are synthetically lethal, demonstrating that Rab5-mediated establishment of endosomal identity is essential for A. nidulans.
Molecular biology of the cell 08/2010; 21(15):2756-69. · 5.98 Impact Factor
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ABSTRACT: We have set up a fast and easy methodology to identify cell-surface proteins in live yeasts. A non-gel proteomic approach was based on a short period of trypsin treatment followed by peptide separation and identification using nano-LC followed by off-line MS/MS. Candida albicans was used as a model organism and proteins involved in cell wall organization, cell rescue, defense, virulence, transport, protein fate and metabolism were identified. This strategy is a powerful tool to study host-pathogen interactions and to look for potential vaccine candidates and drug targets.
Journal of proteomics 02/2010; 73(7):1404-9. · 5.07 Impact Factor
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ABSTRACT: The study of Saccharomyces cerevisiae cell surface proteins was performed because of their important role in cell wall biogenesis and in the physiology of the yeast. Two different proteomic approaches were carried out. First, proteins loosely associated or S-S linked to structural wall components were released by treatment of whole intact cells with dithiothreitol, separated by 2D-PAGE and identified by mass spectrometry. Second, cell surface-exposed proteins (surfome) were digested with trypsin and DTT from whole intact cells, and analyzed by LC-MS/MS. Ninety-nine different proteins were identified: 67 with DTT treatment and 52 with DTT and trypsin digestion. These proteins were classified in different cellular processes: control of cell wall organization, cell rescue, defence, and virulence, protein fate, protein synthesis and metabolism. Most of the proteins have already been reported as present on the cell surface showing that the yeast cell surface is composed not only by typical but also by atypical cell wall proteins. "Bona fide" cell wall proteins were identified by both protocols but a higher number with the non-gel strategy. However, only 20% of the proteins identified were common to both protocols, thus, for a complete knowledge of the cell surface proteome, several strategies have to be used.
Journal of proteomics 02/2010; 73(6):1183-95. · 5.07 Impact Factor
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ABSTRACT: The opportunistic human fungal pathogen Candida albicans causes a wide variety of infections including deep systemic syndromes. The C. albicans plasma membrane is an important interface in the host-pathogen relationship. The plasma membrane proteins mediate a variety of functions, including sensing and signalling to the external environment, in which the glycosylphosphatidylinositol (GPI)-anchored membrane proteins play a crucial role. A subproteomic approach to obtain a global picture of the protein composition of the C. albicans plasma membrane was developed, and different strategies were tested in order to extract the largest number of yeast plasma membrane proteins and GPI-anchored membrane proteins. These methods involved: (i) protoplast generation, (ii) mechanical disruption, (iii) ultracentrifugation in sucrose gradients, and (iv) Na(2)CO(3) treatments. To isolate GPI-anchored proteins two additional steps were performed: two-phase separation and phosphatidylinositol-phospholipase C treatment. After LC-MS/MS analysis using both a MALDI-TOF/TOF and a linear ion trap quadrupole, a total of 214 membrane proteins were identified, including 41 already described as plasma membrane proteins, 20 plasma membrane associated proteins, and 22 proteins with unknown membrane localisation. Bioinformatic analysis revealed that this set of C. albicans membrane proteins is highly enriched in proteins involved in biopolymer biosynthesis or transport processes. Furthermore, after phosphatidylinositol-phospholipase C treatment, 12 GPI-anchored membrane proteins were released and identified; most of them are associated with cell wall beta-glucan synthesis and maintenance or are virulence factors, such as phospholipases or aspartyl proteinases.
Proteomics 10/2009; 9(20):4770-86. · 4.43 Impact Factor
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ABSTRACT: There exist, at present, public web repositories for management and storage of proteomic data and also fungi-specific databases. None of them, however, is focused to the specific research area of fungal pathogens and their interactions with the host, and contains proteomics experimental data. In this context, we present Proteopathogen, a database intended to compile proteomics experimental data and to facilitate storage and access to a range of data which spans proteomics workflows from description of the experimental approaches leading to sample preparation to MS settings and peptides supporting protein identification. Proteopathogen is currently focused on Candida albicans and its interaction with macrophages; however, data from experiments concerning different pathogenic fungi species and other mammalian cells may also be found suitable for inclusion into the database. Proteopathogen is publicly available at http://proteopathogen.dacya.ucm.es
Proteomics 09/2009; 9(20):4664 - 4668. · 4.43 Impact Factor
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ABSTRACT: Fusarium oxysporum is a soilborne fungus that causes vascular wilt disease on a wide range of crops. During initial stages of infection, fungal hyphae attach firmly to roots, penetrate the cortex and colonize xylem vessels. The mechanisms underlying root attachment are poorly understood, although it was previously shown that this process depends on Fmk1, a mitogen-activated protein kinase orthologous to the mating/filamentation mitogen-activated protein kinases Fus3/Kss1 in yeast. We investigated the hypothesis that root adhesion is mediated by fungal cell wall proteins (CWPs). To characterize the cell wall subproteome of F. oxysporum, we performed LC-MS/MS analysis of tryptic digests of purified cell walls obtained from adhesion-inducing conditions, identifying a total of 174 proteins, 19 of which contain a predicted signal peptide and 10 of which have a predicted glycosylphosphatidyl-inositol motif. 2-D DIGE was used to compare four different fractions of CWPs extracted from hyphae of the wild-type strain and the Deltafmk1 mutant. We detected 18 proteins differing significantly in abundance between the two strains. Differential expression of five of these proteins was confirmed by RT-PCR analysis. A significant fraction of the subproteome lacked functional information, highlighting the limitations in the current understanding of CWPs in F. oxysporum.
Proteomics 09/2009; 9(20):4755-69. · 4.43 Impact Factor
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ABSTRACT: The Crc protein is a global translational regulator involved in catabolite repression of catabolic pathways for several non-preferred carbon sources in Pseudomonads when other preferred substrates are present. Using proteomic and transcriptomic approaches, we have analyzed the influence of Crc in cells growing in a complete medium, where amino acids are the main carbon source. Inactivation of the crc gene modified the expression of at least 134 genes. Most of them were involved in the transport and assimilation of amino acids or sugars. This allowed envisioning which amino acids are preferentially used. Crc did not inhibit the pathways for proline, alanine, glutamate, glutamine and histidine. These amino acids are good carbon sources for P. putida. In the case of arginine, lysine, aspartate and asparagine, which can be assimilated through several pathways, Crc favored one particular route, inhibiting other alternatives. Finally, Crc-inhibited genes needed to assimilate valine, isoleucine, leucine, tyrosine, phenylalanine, threonine, glycine and serine, amino acids that provide a less efficient growth. Crc has therefore a key role in coordinating metabolism, controlling the sequential assimilation of amino acids when cells grow in a complete medium. Inactivation of crc reduced growth rate, suggesting that Crc optimizes metabolism.
Proteomics 07/2009; 9(11):2910-28. · 4.43 Impact Factor
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ABSTRACT: Murine macrophages (RAW 264.7) were allowed to interact with heat-inactivated cells of Candida albicans SC5314 during 45 min. The proteomic response of the macrophages was then analyzed using 2-D gel electrophoresis. Many proteins having differential expression with respect to control macrophages were identified, and their functions were related to important processes, such as cytoskeletal organization, signal transduction, metabolism, protein biosynthesis, stress response and protein fate. Several of these proteins have been described as being involved in the process of inflammation, such as Erp29, Hspa9a, AnxaI, Ran GTPase, P4hb, Clic1 and Psma1. The analysis of the consequences of their variation unravels an overall anti-inflammatory response of macrophages during the interaction with heat-inactivated cells. This result was corroborated by the measurement of TNF-alpha and of ERK1/2 phosphorylation levels. This anti-inflammatory effect was contrary to the one observed with live C. albicans cells, which induced higher TNF-alpha secretion and higher ERK1/2 phosphorylation levels with respect to control macrophages.
Proteomics 07/2009; 9(11):2995-3010. · 4.43 Impact Factor
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ABSTRACT: Candida albicans is a human commensal and opportunistic pathogen that participates in biofilm formation on host surfaces and on medical devices. We used DIGE analysis to assess the cytoplasmic and non-covalently attached cell-surface proteins in biofilm formed on polymethylmethacrylate and planktonic yeast cells and hyphae. Of the 1490 proteins spots from cytoplasmic and 580 protein spots from the surface extracts analyzed, 265 and 108 were differentially abundant respectively (>or=1.5-fold, p <0.05). Differences of both greater and lesser abundance were found between biofilms and both planktonic conditions as well as between yeast cells and hyphae. The identity of 114 cytoplasmic and 80 surface protein spots determined represented 73 and 25 unique proteins, respectively. Analyses showed that yeast cells differed most in cytoplasmic profiling while biofilms differed most in surface profiling. Several processes and functions were significantly affected by the differentially abundant cytoplasmic proteins. Particularly noted were many of the enzymes of respiratory and fermentative pentose and glucose metabolism, folate interconversions and proteins associated with oxidative and stress response functions, host response, and multi-organism interaction. The differential abundance of cytoplasmic and surface proteins demonstrated that sessile and planktonic organisms have a unique profile.
Proteomics 04/2009; 9(8):2230-52. · 4.43 Impact Factor
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ABSTRACT: Over the past two decades, mass spectrometry (MS) has ceased to be a fairly exotic technique banished from the protein scientists' mind to become a seminal tool for deciphering the information encoded in the genomes of many biological species. Clues to this shift in the modus operandi for characterizing their proteomes stem from the progressive availability of full genome sequences and well-annotated protein databases of many model (micro)organisms, the development both of soft ionization methods for large biomolecules (peptides and proteins) and of innovative instrumentation designs, and the introduction of sophisticated search algorithms able to correlate MS information with sequence databases, to name but a few. Here we integrate the typical MS-based strategy for identifying proteins of Candida albicans, an opportunistic fungal pathogen of humans, which have proved to be present during systemic infection and targeted by the immune system as a consequence of its interaction with the host (i.e., the C. albicans immunome).
Methods in molecular biology (Clifton, N.J.) 02/2009; 470:187-235.
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ABSTRACT: 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.
Methods in molecular biology (Clifton, N.J.) 02/2009; 470:369-411.
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ABSTRACT: The intracellular trafficking/survival strategies of the opportunistic human pathogen Candida albicans are poorly understood. Here we investigated the infection of RAW264.7 macrophages with a virulent wild-type (WT) filamentous C. albicans strain and a hyphal signalling-defective mutant (efg1Delta/cph1Delta). A comparative analysis of the acquisition by phagosomes of actin, and of early/late endocytic organelles markers of the different fungal strains was performed and related to Candida's survival inside macrophages. Our results show that both fungal strains have evolved a similar mechanism to subvert the 'lysosomal' system, as seen by the inhibition of the phagosome fusion with compartments enriched in the lysobisphosphatidic acid and the vATPase, and thereby the acquisition of a low pH from the outset of infection. Besides, the virulent WT strain displayed additional specific survival strategies to prevent its targeting to compartmentsdisplaying late endosomal/lysosomal features, such as induction of active recycling out of phagosomes of the lysosomal membrane protein LAMP-1, the lysosomal protease cathepsin D and preinternalized colloidal gold. Finally, both virulent and efg1Delta/cph1Delta mutant fungal strains actively suppressed the production of macrophage nitric oxide (NO), although their cell wall extracts were potent inducers of NO.
Cellular Microbiology 01/2009; 11(4):560-89. · 5.46 Impact Factor
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ABSTRACT: 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.
Proteomics 08/2008; 8(13):2651-64. · 4.43 Impact Factor
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ABSTRACT: 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.
Proteomics. Clinical applications 04/2008; 2(4):596-618. · 1.97 Impact Factor
