Biomedical subjects

D Gozalbo

Publications and source records attributed to D Gozalbo.

At least 19 recordsLinked to original sources

PubMed2006

Toll-like receptor 4 defective mice carrying point or null mutations do not show increased susceptibility to Candida albicans in a model of hematogenously disseminated infection.

We have studied the role of TLR4 in murine defenses against Candida albicans in two TLR4-defective mouse strains: C3H/HeJ mice which have defective TLR4 signaling, and TLR4-/- knockout mice. Both TLR4-defective mice strains experimentally infected with virulent C. albicans cells showed no significant difference in survival as compared with their respective controls. Recruitment of neutrophils to the peritoneal cavity of i.p. infected mice was not affected in TLR4-/-animals, but significantly enhanced in C3H/HeJ mice, compared with their control mice. In vitro production of TNF-alpha by macrophages from both types of TLR4-defective mice, in response to yeasts and hyphae of C. albicans, was not diminished as compared with production by macrophages from wild-type mice. In vitro production of TNF-alpha by yeast-stimulated splenocytes from mice intravenously infected with the low-virulence C. albicans PCA2 strain was not affected in TLR4-defective mice, but the TNF-alpha production in response to hyphae was higher in TLR4-defective than in control animals; the production of IFN-gamma by these splenocytes was similar to controls, as well as the frequency of IFN-gamma-producing CD4+T lymphocytes, indicating that TLR4-defective mice are capable of mounting a Th1 adaptive immune response. Our data indicate that TLR4 is dispensable for murine immune resistance to C. albicans.

Animals↗

PubMed2002

Candida albicans UBI3 and UBI4 promoter regions confer differential regulation of invertase production to saccharomyces cerevisiae cells in response to stress.

Candida albicans ubiquitin genes UBI3 and UBI4 encode a ubiquitin-hybrid protein involved in ribosome biogenesis and polyubiquitin, respectively. In this work we show that UBI3 and UBI4 promoter regions confer differential expression consistent with the function of their encoded gene products. Hybrid genes were constructed containing the SUC2 coding region under the control of UBI3 or UBI4 promoters in the yeast vector pLC7. Invertase production in Saccharomyces cerevisiae transformants was differentially regulated: the UBI4 promoter was induced by stress conditions (thermal upshift and/or starvation) whereas the UBI3 promoter conferred constitutive invertase production in growing yeast cells. These results indicate that the UBI4 promoter is regulated by stress-response signaling pathways, whereas the UBI3 promoter is controlled according to the requirement for protein synthesis to support cell growth.

Candida albicans↗

PubMed2001

The Candida albicans cell wall-associated glyceraldehyde-3-phosphate dehydrogenase activity increases in response to starvation and temperature upshift.

We have determined the effect of environmental factors (mild thermal upshift and starvation) on the Candida albicans cell wall-associated glyceraldehyde-3-phosphate dehydrogenase (cwGAPDH) activity. Temperature upshift (from 28 to 37 degrees C) and/ or starvation (at 28 or 37 degrees C in water) of exponentially growing yeast cells caused an increase in cwGAPDH activity (3 to 5-, and 7 to 8-fold, respectively). This increase in activity did not correlate with an increase in the amount of cwGAPDH protein present, as determined by flow cytometry, immunoelectron microscopy and Western-blotting. These results indicate that thermal upshift and starvation cause an activation of the cwGAPDH in C. albicans cells.

Candida albicans↗

PubMed2000

Molecular cloning and characterization of the Candida albicans UBI3 gene coding for a ubiquitin-hybrid protein.

Using a polyubiquitin cDNA as a probe, we have isolated a clone (pPR3, a pEMBLYe23 derivative plasmid) containing the Candida albicans UBI3 gene coding for a fusion protein. This protein is formed by one ubiquitin subunit fused, at its C-terminus, to an unrelated peptide which is similar to the ribosomal protein encoded by the 3' tail of the Saccharomyces cerevisiae UBI3 gene. Southern blot analysis of chromosomal DNA probed with the 3' non-ubiquitin tail of UBI3 indicated that only one homologous gene is present in the C. albicans genome. Heterelogous expression of pPR3 in a S. cerevisiae ubi3 mutant strain complements the mutant phenotype (slow growth) conferred by the ubi3 defect; this provides direct evidence indicating that the clone contains the C. albicans UBI3 gene Northern blot analysis showed that UBI3 gene is expressed in yeast and germ-tube cells of C. albicans, although the UBI3 mRNA levels in starved yeast cells are below the detection limit; UBI3 mRNA drops to undetectable levels on shifting the temperature of growing yeast cells from 28 degrees C to 42 degrees C. When Northern blot analysis was performed using a specific probe for the polyubiquitin (UBI4) gene, no drop in the mRNA levels was detected following thermal upshift or in starved cells. These results indicate that stress conditions (starvation or thermal upshift) negatively regulate UBI3 expression (transcriptional arrest and/or enhanced mRNA decay), and suggest that UBI4 gene provides ubiquitin during the stress response. In addition, we failed to obtain C. albicans UBI3 null mutant cells by sequential disruption of both alleles using the hisG::URA3::hisG ('ura-blaster') cassette, suggesting that null mutants cells may be unable to grow on selective media after transformation.

Amino Acid Sequence↗

PubMed2000

Complementation of Saccharomyces cerevisiae mutations in genes involved in translation and protein folding (EFB1 and SSB1) with Candida albicans cloned genes.

We have demonstrated that the expression of Candida albicans genes involved in translation and protein folding (EFB1 and SSB1) complements the phenotype of Saccharomyces cerevisiae mutants. The elongation factor 1beta (EF-1beta) is essential for growth and efb1 S. cerevisiae null mutant cells are not viable; however, viable haploid cells, carrying the disrupted chromosomal allele of the S. cerevisiae EFB1 gene and pEFB1, were isolated upon sporulation of a diploid strain which was heterozygous at the EFB1 locus and transformed with pEFB1 (a pEMBLYe23 derivative plasmid containing an 8-kb DNA fragment from the C. albicans genome which contains the EFB1 gene). This indicates that the C. albicans EFB1 gene encodes a functional EF-1beta. Expression of the SSB1 gene from C. albicans, which codes for a member of the 70-kDa heat shock protein family, in S. cerevisiae ssb1 ssb2 double mutant complements the mutant phenotype (poor growth particularly at low temperature, and sensitivity to certain protein synthesis inhibitors, such as paromomycin). This complementation indicates that C. albicans Ssbl may function as a molecular chaperone on the translating ribosomes, as described in S. cerevisiae. Northern blot analysis showed that SSB mRNA levels increased after mild cold shift (28 degrees C to 23 degrees C) and rapidly decreased after mild heat shift (from 28 degrees C to 37 degrees C, and particularly to 42 degrees C), indicating that SSB1 expression is regulated by temperature. Therefore, Ssb1 may be considered as a molecular chaperone whose pattern of expression is similar to that found in ribosomal proteins, according to its common role in translation.

Candida albicans↗

PubMed2000

Identification of Candida albicans clinical isolates by PCR amplification of an EFB1 gene fragment containing an intron-interrupted open reading frame.

The use of a single pair of primers, deduced from the intron and exon nucleotide sequences of the Candida albicans EFB1 gene, in polymerase chain reaction (PCR) assays performed with whole cells of both laboratory strains and clinical isolates of Candida species, resulted in the species-specific amplification of a 785 bp DNA fragment in C. albicans strains. Clinical C. albicans isolates were tested, and 85 out of 86 generated the expected PCR-amplified product; other Candida species, both laboratory strains and clinical isolates, as well as laboratory strains belonging to other fungal genera, including medically relevant taxa, failed to amplify any DNA fragment. In addition, unusual C. albicans isolates (glucosamine- and N-acetylglucosamine-negative) from Africa also yielded the expected PCR-generated DNA fragment. These results indicate that genes containing intron sequences may be useful to design species-specific primers for the identification of fungal strains by PCR.

Candida albicans↗

PubMed1999

Purification of a biologically active recombinant glyceraldehyde 3-phosphate dehydrogenase from Candida albicans.

We report here the purification of a functionally active recombinant glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Candida albicans. The GAPDH protein encoded by the TDH1 gene was obtained as a glutathione S-transferase fusion protein by expression in the vector pGEX-4T-3, and purified by affinity chromatography and thrombin digestion. The purified protein displays GAPDH enzymatic activity (42 micromol NADH min(-1) mg(-1)) as well as the laminin and fibronectin binding activities previously described. In addition, the recombinant GAPDH is covalently modified by NAD linkage; this modification is stimulated by nitric oxide and probably involves a sulfhydryl group (cysteine) residue since it is inhibited by Hg(2+) and cysteine.

Candida albicans↗

PubMed1999

Clinical strains of Candida albicans express the surface antigen glyceraldehyde 3-phosphate dehydrogenase in vitro and in infected tissues.

We have previously described the presence of an enzymatically active form of glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) in the cell surface of Candida albicans ATCC 26555 which is also a fibronectin and laminin binding protein. Immunohistochemical analysis of tissue sections from patients with disseminated candidiasis with a polyclonal antiserum to GAPDH from C. albicans (PAb anti-CA-GAPDH) revealed that the enzyme is expressed at the surface of fungal cells in infected tissues. The same PAb detected the presence of GAPDH species, with a molecular mass of approximately 33 kDa, in cell wall extracts obtained from clinical isolates of the fungus. These cell surface-bound GAPDH moieties exhibited a dose-dependent dehydrogenase activity. These results indicate that this cell surface-bound GAPDH plays a role during infection probably contributing to the attachment of fungal cells to host tissues.

Antigens, Fungal↗

PubMed1998

Molecular cloning and characterization of a Candida albicans gene coding for cytochrome c haem lyase and a cell wall-related protein.

Immunoscreening of a Candida albicans cDNA library with a monoclonal antibody (mAb 4C12) recognizing an epitope present in high-molecular-weight mannoprotein (HMWM) components specific for the mycelial cell walls (a 180 kDa component and a poly-dispersed 260 kDa species) resulted in the isolation of the gene CaCYC3 encoding for cytochrome c haem lyase (CCHL). The CaCYC3 gene was transcribed preferentially in mycelial cells in which two mRNA transcripts of 0.8 and 1 kb were found. The nucleotide and the deduced amino acid sequences of this gene displayed 45% homology and 46% identity, respectively, to the Saccharomyces cerevisiae CYC3 gene and shared common features with other reported genes encoding for CCHL. The CaCYC3 gene restored the respiratory activity when transformed in a S. cerevisiae cyc3- mutant strain. A C. albicans CYC3 null mutant was constructed after sequential disruption using the hisG::URA3::hisG ('ura-blaster') cassette. Null mutant cells were unable to use lactate as a sole carbon source and had a reduced ability to form germ tubes. Western immunoblotting analysis of subcellular fractions from wild-type and null mutant strains demonstrated the presence of two gene products, a 33kDa mitochondrial protein and a 40 kDa cell wall-associated moiety reacting with antibodies against CCHL, in both yeast cells and germ tubes. mAb 4C12 still reacted with the CaCYC3 null mutant (by immunofluorescence and immunoblotting) but showed an altered pattern of immunoreactivity against cell wall HMWM species, indicating a relationship between these moieties and the CaCYC3 gene products. The results suggest that the CaCYC3 gene encodes two proteins, one targeted to the mitochondria and the other to the cell wall.

Amino Acid Sequence↗

PubMed1998

The cell wall-associated glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is also a fibronectin and laminin binding protein.

By immunoelectron microscopy with a polyclonal antibody against the cytosolic glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Candida albicans (anti-GAPDH PAb), the protein was clearly detected at the outer surface of the cell wall, particularly on blastoconidia, as well as in the cytoplasm. Intact blastoconidia were able to adhere to fibronectin and laminin immobilized on microtiter plates, and this adhesion was markedly reduced by both the anti-GAPDH PAb and soluble GAPDH from Saccharomyces cerevisiae. In addition, semiquantitative flow cytometry analysis with the anti-GAPDH PAb showed a decrease in antibody binding to cells in the presence of soluble fibronectin and laminin. Purified cytosolic C. albicans GAPDH was found to bind to fibronectin and laminin in a ligand Western blot assay. These observations suggest that the cell wall-associated form of the GAPDH in C. albicans could be involved in mediating adhesion of fungal cells to fibronectin and laminin, thus contributing to the attachment of the microorganism to host tissues and to the dissemination of Candida infection.

Candida albicans↗

PubMed1998

Cell wall and secreted proteins of Candida albicans: identification, function, and expression.

The cell wall is essential to nearly every aspect of the biology and pathogenicity of Candida albicans. Although it was initially considered an almost inert cellular structure that protected the protoplast against osmotic offense, more recent studies have demonstrated that it is a dynamic organelle. The major components of the cell wall are glucan and chitin, which are associated with structural rigidity, and mannoproteins. The protein component, including both mannoprotein and nonmannoproteins, comprises some 40 or more moieties. Wall proteins may differ in their expression, secretion, or topological location within the wall structure. Proteins may be modified by glycosylation (primarily addition of mannose residues), phosphorylation, and ubiquitination. Among the secreted enzymes are those that are postulated to have substrates within the cell wall and those that find substrates in the extracellular environment. Cell wall proteins have been implicated in adhesion to host tissues and ligands. Fibrinogen, complement fragments, and several extracellular matrix components are among the host proteins bound by cell wall proteins. Proteins related to the hsp70 and hsp90 families of conserved stress proteins and some glycolytic enzyme proteins are also found in the cell wall, apparently as bona fide components. In addition, the expression of some proteins is associated with the morphological growth form of the fungus and may play a role in morphogenesis. Finally, surface mannoproteins are strong immunogens that trigger and modulate the host immune response during candidiasis.

Candida albicans↗

PubMed1997

Cloning of a cDNA fragment encoding part of the protein moiety of the 58-kDa fibrinogen-binding mannoprotein of Candida albicans.

Immunoscreening of a Candida albicans expression library with antibodies against the 58 kDa fibrinogen-binding mannoprotein (mp58) of the fungus resulted in the isolation of clones encoding the protein moiety of this molecule. Sequence of the 0.9 kb cDNA of one of the clones selected for further analysis, revealed an open reading frame coding for 292 amino acids, which displays sequence similarity to proteins belonging to a family of immunodominant antigens of Aspergillus spp. The gene corresponding to this cDNA was named FBP1 (fibrinogen-binding protein). These results represent the first report on the identification of C. albicans genes encoding surface receptors for host proteins.

Amino Acid Sequence↗

PubMed1997

Molecular cloning of a Candida albicans gene (SSB1) coding for a protein related to the Hsp70 family.

We have cloned and sequenced a Candida albicans gene (SSB1) encoding a potential member of the heat-shock protein seventy (hsp70) family. The protein encoded by this gene contains 613 amino acids and shows a high degree (85%) of sequence identity to the ssb subfamily (ssb1 and ssb2) of the Saccharomyces cerevisiae hsp70 family. The transcribed mRNA (2.1 kb) is present in similar amounts both in yeast and germ tube cells of C. albicans.

Amino Acid Sequence↗

PubMed1997

Hemin induces germ tube formation in Candida albicans.

Hemin induced germination of Candida albicans blastoconidia when cells grown up to the early exponential phase were shifted from 28 to 37 degrees C (70 to 75% of cells exhibited germ tubes). N-Acetyl-D-glucosamine (GlcNAc), another inducer of myceliation in this fungus, caused a similar effect. The combination of hemin and GlcNAc resulted in a higher percentage (95%) of blastoconidial germination. These results suggest that in addition to temperature, hemin levels and carbon source may coordinately regulate the expression of subsets of genes involved in the yeast-to-mycelium transition in C. albicans.

Acetylglucosamine↗

PubMed1997

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is a surface antigen.

A lambda gt11 cDNA library from Candida albicans ATCC 26555 was screened by using pooled sera from two patients with systemic candidiasis and five neutropenic patients with high levels of anti-C. albicans immunoglobulin M antibodies. Seven clones were isolated from 60,000 recombinant phages. The most reactive one contained a 0.9-kb cDNA encoding a polypeptide immunoreactive only with sera from patients with systemic candidiasis. The whole gene was isolated from a genomic library by using the cDNA as a probe. The nucleotide sequence of the coding region showed homology (78 to 79%) to the Saccharomyces cerevisiae TDH1 to TDH3 genes coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and their amino acid sequences showed 76% identity; thus, this gene has been named C. albicans TDH1. A rabbit polyclonal antiserum against the purified cytosolic C. albicans GAPDH (polyclonal antibody [PAb] anti-CA-GAPDH) was used to identify the GAPDH in the beta-mercaptoethanol extracts containing cell wall moieties. Indirect immunofluorescence demonstrated the presence of GAPDH at the C. albicans cell surface, particularly on the blastoconidia. Semiquantitative flow cytometry analysis showed the sensitivity of this GAPDH form to trypsin and its resistance to be removed with 2 M NaCl or 2% sodium dodecyl sulfate. The decrease in fluorescence in the presence of soluble GAPDH indicates the specificity of the labelling. In addition, a dose-dependent GAPDH enzymatic activity was detected in intact blastoconidia and germ tube cells. This activity was reduced by pretreatment of the cells with trypsin, formaldehyde, and PAb anti-CA-GAPDH. These observations indicate that an immunogenic, enzymatically active cell wall-associated form of the glycolytic enzyme GAPDH is found at the cell surface of C. albicans cells.

Antibodies, Fungal↗

PubMed1996

Molecular cloning and characterization of a Candida albicans gene (EFB1) coding for the elongation factor EF-1 beta.

A Candida albicans gene homologous to Saccharomyces cerevisiae elongation factor 1 beta was isolated by screening a genomic DNA library using a C. albicans cDNA as a probe. This cDNA was previously obtained by immunoscreening of an expression library with polyclonal antibodies raised against candidal cell wall components. Sequence analysis of the cDNA and the whole C. albicans gene (EMBL accession number X96517) revealed an intron-interrupted open reading frame of 639 base pairs that encodes a 213 amino acid protein. Exon sequences are highly homologous (74%) to S. cerevisiae EFB1, whereas intron sequence is less conserved (34% identity), and the predicted amino acid sequence shares about 73% identity.

Animals↗

PubMed1996

Ubiquitin-like epitopes associated with Candida albicans cell surface receptors.

We have recently reported the cloning of a Candida albicans polyubiquitin gene and the presence of ubiquitin in the cell wall of this fungus. The polyubiquitin cDNA clone was isolated because of its reactivity with antibodies generated against the candidal 37-kDa laminin-binding protein. In the present study, we have further investigated the relationship between ubiquitin and cell wall components displaying receptor-like activities, including the 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. Two-dimensional electrophoretic analysis and immunoblot experiments with antibodies against ubiquitin and the individually purified receptor-like molecules confirmed that these cell surface components are ubiquitinated. In an enzyme-linked immunosorbent assay, polyclonal antisera to each receptor reacted with ubiquitin, thus demonstrating that the purified receptor preparations used as immunogens contained ubiquitin-like epitopes. It is proposed that ubiquitin may play a role in modulating the activity of these receptors and in the interaction of C. albicans cells with host structures.

Animals↗

PubMed1996

A Candida albicans gene expressed in Saccharomyces cerevisiae results in a distinct pattern of mRNA processing.

Two plasmids (derived from YCplac22 and YEplac112) carrying a Candida albicans gene (including the 5' non-coding promoter sequences) coding for a 30 kDa membrane-bound protein, were used to transform Saccharomyces cerevisiae cells. A 30 kDa protein was immunodetected by Western blot in the membrane fraction of transformants. Northern analysis showed the presence of three mRNA species (of about 1.1, 0.7 and 0.5 kb) hybridizing with the C. albicans gene as a probe. The same result was obtained using the 5' and 3' regions of the gene as probes, whereas only a 1.1 kb mRNA was found in C. albicans and none was detected in S. cerevisiae control transformants. Thus, heterologous expression of this gene in S. cerevisiae results in a distinct pattern of mRNA processing, either due to the location on plasmid vectors and/or to differences in the mRNA processing systems in the two microorganisms.

Candida albicans↗