Biomedical subjects

Concha Gil

Publications and source records attributed to Concha Gil.

22 records · Page 2Linked to original sources

PubMed2004

The GPI-anchored protein CaEcm33p is required for cell wall integrity, morphogenesis and virulence in Candida albicans.

Ecm33p is a widely distributed fungal protein with functional relevance, clearly demonstrated by ecm33Delta mutant phenotypes, mainly related to the cell wall. Homology searches with Saccharomyces cerevisiae genes identified Candida albicans Ecm33p, as well as the two other proteins of its family: Pst1p and the product of YCL048w. C. albicans Ecm33p is a 423 aa protein which has the typical features of cell-surface GPI proteins and is able to complement S. cerevisiae ecm33Delta cell wall defects. Heterozygous (RML1) and homozygous (RML2) mutants of CaECM33 were obtained, as well as a single and a double reintegrant (RML3 and RML4, respectively). Caecm33 mutant strains displayed an aberrant morphology, being more rounded and bigger than the wild-type, suggesting morphogenetic defects. They also exhibited cell wall defects, with enhanced sensitivity to different compounds that interfere in polymerization of cell wall components (Calcofluor white, Congo red and hygromycin B) and a marked tendency to flocculate extensively. In addition, CaEcm33p is required for normal C. albicans yeast-to-hyphae transition in vitro. In liquid medium (5 % serum), the transition was delayed in Caecm33 mutants, and after 24 h the culture contained very abnormal large and rounded cells. On solid medium (10 % serum, Spider or SLADH) RML2 failed to produce hyphae and media invasiveness. CaECM33 showed a gene dosage effect, demonstrated by the intermediate phenotype of the heterozygous mutants RML1 and confirmed by Northern blot analysis. Furthermore, CaEcm33p is also involved in C. albicans virulence. In a murine systemic model of infection, 100 % mouse survival and no kidney or brain colonization were obtained 30 days after infection with 10(6) Candida cells of any homozygous or heterozygous Caecm33Delta mutant tested. In contrast, all mice infected with parental or RML4 (two CaECM33 copies reintegrated) strains died in a few days, showing that, in these conditions, two CaECM33 copies were required for virulence.

Animals↗

PubMed2003

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.

Candida albicans↗

PubMed2003

Analysis of the Candida albicans proteome. II. Protein information technology on the Net (update 2002).

Candida albicans is an important fungal model organism of noteworthy clinical interest in modern medicine. Different initiatives addressing its sequencing and physical mapping have been carried out. The C. albicans genome sequence is currently near to completion at Stanford University, heralding new challenges in proteomic research and functional analyses of its gene products. This review presents an update of the most relevant data resources that are available through the World Wide Web to scientists working in the area of the analysis of the C. albicans proteome. An overview of the current status of the main universal protein sequence databases and specialized data collections for C. albicans is given. Various issues of the single public C. albicans 2D-PAGE database are also described, highlighting the significance of setting up graphical query interface-based databanks to visualize 2D-PAGE images through the Net. Finally, we also emphasize the pressing need to create a "cyber-bioknowledge library" that will integrate all the databases developed at the different levels for the understanding of life processes as well as bioinformatic tools for interpreting this deluge of data generated through the Internet.

Candida albicans↗

PubMed2002

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.

Blotting, Western↗