Biomedical subjects

Rosalía Diez-Orejas

Publications and source records attributed to Rosalía Diez-Orejas.

4 recordsLinked to original sources

PubMed2006

Integrated proteomics and genomics strategies bring new insight into Candida albicans response upon macrophage interaction.

The interaction of Candida albicans with macrophages is considered a crucial step in the development of an adequate immune response in systemic candidiasis. An in vitro model of phagocytosis that includes a differential staining procedure to discriminate between internalized and non-internalized yeast was developed. Upon optimization of a protocol to obtain an enriched population of ingested yeasts, a thorough genomics and proteomics analysis was carried out on these cells. Both proteins and mRNA were obtained from the same sample and analyzed in parallel. The combination of two-dimensional PAGE with MS revealed a total of 132 differentially expressed yeast protein species upon macrophage interaction. Among these species, 67 unique proteins were identified. This is the first time that a proteomics approach has been used to study C. albicans-macrophage interaction. We provide evidence of a rapid protein response of the fungus to adapt to the new environment inside the phagosome by changing the expression of proteins belonging to different pathways. The clear down-regulation of the carbon-compound metabolism, plus the up-regulation of lipid, fatty acid, glyoxylate, and tricarboxylic acid cycles, indicates that yeast shifts to a starvation mode. There is an important activation of the degradation and detoxification protein machinery. The complementary genomics approach led to the detection of specific pathways related to the virulence of Candida. Network analyses allowed us to generate a hypothetical model of Candida cell death after macrophage interaction, highlighting the interconnection between actin cytoskeleton, mitochondria, and autophagy in the regulation of apoptosis. In conclusion, the combination of genomics, proteomics, and network analyses is a powerful strategy to better understand the complex host-pathogen interactions.

Animals↗

PubMed2004

Two-dimensional reference map of Candida albicans hyphal forms.

We have set up an in vitro model of culture of Candida albicans fungal cells that mimics the physiological conditions found in human beings in order to carry out studies of host-pathogen interaction. Under these conditions, C. albicans undergoes dimorphic transition (yeast-hyphae); this morphological change has been proposed as a virulence factor for this fungus. C. albicans cytoplasmic extract from hyphae cells was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and a reference map was obtained. Protein identification was carried out by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) or a MALDI-TOF/TOF mass spectrometer. A total of 106 spots, excised from 2-D gels, were analyzed. This resulted in the identification of 43 proteins involved in metabolism, 13 involved in transcription, protein synthesis, and fate, 8 involved in cell rescue, virulence, and defense, and 2 proteins of unknown function. This reference map is an important tool for future studies of protein differential expression after host-pathogen interaction.

Candida albicans↗

PubMed2004

Contribution of the antibodies response induced by a low virulent Candida albicans strain in protection against systemic candidiasis.

A low virulent Candida albicans mutant, CNC13, deleted in the Mitogen Activated Protein (MAP) kynase HOG1 was used to immunize BALB/c mice. Hog1p is essential for the oxidative stress and hyperosmolarity responses. Several doses and immunization procedures were employed. The protection capacity of the different sera generated was analyzed in a murine model of systemic candidiasis. Using a proteomic approach (two-dimensional gel electrophoresis followed by Western blotting), we were able to distinguish two categories of serum: protective and nonprotective, which showed different titres of total Immunoglobulins (Igs) and IgG2a (analyzed by enzyme-linked immunosorbent assay). The levels of Igs and IgG2a in protective sera were significantly higher compared to nonprotective sera. The pattern of a "nonprotective" profile was composed of enolase (Eno1p), transketolase, heat shock protein and methionine synthase. Only antibodies against enolase are the IgG2a isotype. The pattern of a "protective" sera, on the other hand, was composed of antibodies against the following antigens: several isoforms of Eno1p, pyruvate decarboxylase, pyruvate kynase, a protein of the 40S ribosomal subunit, triosephosphate isomerase, DL-glycerol phosphatase and fructose-bisphosphate aldolase. All these antibodies are the IgG2a isotype. The proteins described in the protective sera might be useful for future vaccine development.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2004

Low virulent strains of Candida albicans: unravelling the antigens for a future vaccine.

Several low virulent Candida albicans mutant strains: CM1613 (deleted in the Mitogen Activated Protein (MAP) Kinase MKC1), CNC13 (deleted in the MAP-kinase HOG1) and the morphological mutant 92' were used as vaccines employing a murine model of systemic candidiasis. In this vaccination trial, only the CNC13 strain was able to induce protection against a subsequent infection with a lethal dose of the wild-type strain. The protection induced by CNC13 vaccinated animals resulted in 60-70% percent of survival. These results demonstrate that collaboration between cellular and humoral responses, induced by the CNC13 mutant, elicited a long lasting and effective protection. Using a proteomic approach (two-dimensional gel electrophoresis followed by Western blotting), twenty-five C. albicans immunogenic proteins were detected and identified by matrix-assisted laser desorption/ionization and/or tandem mass spectrometry. We were able to define an antibody pattern in the sera from the nonvaccinating strains (92' and CM1613), which was different from the profile detected in the sera from surviving animals (vaccinated with the CNC13 mutant). The utility of this proteomic approach has allowed us to identify antigens that induce protective IgG2a antibody isotype in the sera from vaccinated animals: enolase (Eno1p), pyruvate kinase (Cdc19p), pyruvate decarboxylase (Pdc11p), a component from the 40S ribosomal subunit (Bel1p), triosephosphate isomerase (Tpi1p), DL-glycerol phosphatase (Rhr2p), fructose-bisphosphate aldolase (Fba1p) and two new protective antigens: IMP dehydrogenase (Imh3p), and acetyl-CoA synthetase (Acs2p). The antigenic proteins that promote protective antibodies described in this work are excellent candidates for a future fungal vaccine; their heterologous expression and vaccine design is currently underway.

Animals↗