Amelia Murgui

Dimorphism in Candida albicans: contribution of mannoproteins to the architecture of yeast and mycelial cell walls

Wall mannoproteins of the two (yeast and mycelial) cellular forms of Candida albicans were solubilized by different agents. Boiling in 2% (w/v) SDS was the best method, as more than 70% of the total mannoprotein was extracted. Over 40 different bands (from 15 to 80 kDal) were detected on SDS-polyacrylamide gel electrophoresis of this material. The residual wall mannoproteins were released after enzymic (Zymolyase and endogenous wall beta-glucanases) degradation of wall glucan, suggesting that they are covalently linked to this structural polymer. Four bands (of 160 kDal, 205 kDal and higher molecular mass) were observed in the material released from yeast walls but only the two smaller components were detected in the material obtained from mycelial walls. Moreover, the mannoproteins of high molecular mass, which are covalently linked in walls of normal cells, were not incorporated into walls of regenerating protoplasts, but non-covalently linked mannoproteins were retained from the beginning of the process.

Formation of a New Cell Wall by Protoplasts of Candida albicans: Effect of Papulacandin B, Tunicamycin and Nikkomycin

Incorporation of polysaccharides into the walls of regenerating protoplasts of Candida albicans was followed in the presence of papulacandin B, tunicamycin and nikkomycin. With the first drug, chitin was incorporated normally whereas incorporation of glucans and mannoproteins was significantly decreased. Tunicamycin decreased incorporation of all wall polymers when added at the beginning of the regeneration process but blocked only mannan and alkali-insoluble glucan incorporation when added after 5 h. Nikkomycin inhibited chitin synthesis, and the walls formed by the protoplasts were enriched in alkali-soluble glucan. Pulse-chase experiments suggested that a precursor-product relationship between the alkali-soluble and alkali-insoluble glucans existed in the wall. The results obtained with the antibiotics were confirmed and extended by cytological studies using wheat-germ agglutinin labelled with colloidal gold and concanavalin A-ferritin as specific markers of chitin and mannoproteins respectively. The results support the idea that regeneration of walls by protoplasts occurs in two steps: firstly, a chitin microfibrillar skeleton is formed, and in a later step glucan-mannoprotein complexes are added to the growing structure. The chitin skeleton probably allows the orderly spatial arrangement of the other polymers giving rise to the regenerated cell wall.

Effect of papulacandin B and calcofluor white on the incorporation of mannoproteins in the wall of Candida albicans blastospores

Incorporation of mannoproteins into the walls of Candida albicans blastospores (yeast phase) was followed by continuous labelling and pulse-chase experiments. The effect in the process of compounds that interfere with synthesis (papulacandin B) or assembly (calcofluor white) of structural polymers was also assessed. Mannoproteins which are kept in place by non-covalent bonds (mainly hydrogen bonds) were incorporated rapidly after their release into the periplasmic space, this process being blocked by calcofluor white. The stain had no effect on the incorporation of covalently linked mannoproteins. Papulacandin B inhibited formation of beta-glucans and incorporation of covalently linked mannoprotein molecules, whereas incorporation of hydrogen-bonded species took place normally. The results suggest that the formation of the non-covalent bonds between the mannoproteins occurs once they are secreted into the periplasmic space, whereas the formation of covalent connections between mannoproteins and wall glucan takes place at the level of the plasma membrane.

Different adhesins for type IV collagen on Candida albicans: identification of a lectin-like adhesin recognizing the 7S(IV) domain

Adherence of the opportunistic pathogen Candida albicans to basement membrane (BM) proteins is considered a crucial step in the development of candidiasis. In this study the interactions of C. albicans yeast cells with the three main domains of type IV collagen, a major BM glycoprotein, were analysed. C. albicans adhered to the three immobilized domains by different mechanisms. Adhesion to the N-terminal cross-linking domain (7S) required the presence of divalent cations, whereas interaction with the central collagenous domain (CC) was cation-independent. Recognition of the C-terminal non-collagenous domain (NC1) was partially cation-dependent. Binding inhibition assays with the corresponding domains in soluble form showed that these interactions were specific. Both Ca(2+) and Mg(2+) promoted adhesion to the 7S domain and the interaction was completely abolished by EDTA. Treatment of the 7S domain, or its subunits, with N-glycosidase F reduced yeast binding by approximately 70%. Moreover, several sugars known to be part of the N-linked oligosaccharide chains of collagen IV inhibited adhesion to immobilized 7S; N-acetylglucosamine, L-fucose and methylmannoside caused a similar inhibition whereas N-acetyllactosamine was a more effective inhibitor. In contrast, glucose, galactose, lactose or heparan sulfate did not affect yeast binding. Combinations of the inhibitory sugars at suboptimal inhibition concentrations did not reduce C. albicans adhesion more than the individual sugars, pointing to a single lectin as responsible for the interaction. These results taken together show that C. albicans utilizes several adhesins for interacting with type IV collagen, and that at least one of them is a lectin which recognizes the 7S(IV) oligosaccharide residues as its receptor.

Some biological features of Candida albicans mutants for genes coding fungal proteins containing the CFEM domain.

Several biological features of Candida albicans genes (PGA10, RBT5 and CSA1) coding for putative polypeptide species belonging to a subset of fungal proteins containing an eight-cysteine domain referred as common in several fungal extracellular membrane (CFEM) are described. The deletion of these genes resulted in a cascade of pleiotropic effects. Thus, mutant strains exhibited higher cell surface hydrophobicity levels and an increased ability to bind to inert or biological substrates. Confocal scanning laser microscopy using concanavalin A-Alexafluor 488 (which binds to mannose and glucose residues) and FUN-1 (a cytoplasmic fluorescent probe for cell viability) dyes showed that mutant strains formed thinner and more fragile biofilms. These apparently contained lower quantities of extracellular matrix material and less metabolically active cells than their parental strain counterpart, although the relative percentage of mycelial forms was similar in all cases. The cell surface of C. albicans strains harbouring deletions for genes coding CFEM-domain proteins appeared to be severely altered according to atomic force microscopy observations. Assessment of the relative gene expression within individual C. albicans cells revealed that CFEM-coding genes were upregulated in mycelium, although these genes were shown not to affect virulence in animal models. Overall, this study has demonstrated that CFEM domain protein-encoding genes are pleiotropic, influencing cell surface characteristics and biofilm formation.

ABG1, a Novel and Essential Candida albicans Gene Encoding a Vacuolar Protein Involved in Cytokinesis and Hyphal Branching

ABSTRACT Immunoscreening of a Candida albicans expression library resulted in the isolation of a novel gene encoding a 32.9-kDa polypeptide (288 amino acids), with 27.7% homology to the product of Saccharomyces cerevisiae YGR106c, a putative vacuolar protein. Heterozygous mutants in this gene displayed an altered budding growth pattern, characterized by the formation of chains of buds, decreasingly in size towards the apex, without separation of the daughter buds. Consequently, this gene was designated ABG1. A conditional mutant for ABG1 with the remaining allele under the control of the MET3 promoter did not grow in the presence of methionine and cysteine, demonstrating that ABG1 was essential for viability. Western analysis revealed the presence of a major 32.9-kDa band, mainly in a particulate fraction (P40) enriched in vacuoles, and tagging with green fluorescent protein confirmed that Abg1p localized to the vacuole. Vacuole inheritance has been linked to the regulation of branching frequency in C. albicans. Under repressing conditions, the conditional mutant had an increased frequency of branching under hyphal inducing conditions and an altered sensitivity to substances that interfered with cell wall assembly. Repression of ABG1 in the conditional mutant strain caused disturbance of normal size and number of vacuoles both in yeast and mycelial cells and also in the asymmetric vacuole inheritance associated with the characteristic pattern of germ tubes and branching in C. albicans. These observations indicate that ABG1 plays a key role in vacuole biogenesis, cytokinesis, and hyphal branching.

CCL27–CCR10 and CXCL12–CXCR4 chemokine ligand-receptor mRNA expression ratio: new predictive factors of tumor progression in cutaneous malignant melanoma

CXCR4, CCR7 and CCR10 chemokine receptors are known to be involved in melanoma metastasis. Our goal was to compare the relative intratumoral mRNA expression of these receptors with that of their corresponding chemokine ligands, CXCL12, CCL19, CCL21, and CCL27 across the full spectrum of human melanoma progression: thin and thick primary melanomas, as well as “in transit”, lymph node, and distant metastases. Expression was quantified by real-time RT-PCR in 103 melanoma samples: 51 primary tumors and 52 metastases. Particular emphasis was focused on chemokine ligand-receptor expression ratios. Immunohistochemistry was performed to identify the cell types expressing these molecules. CXCL12–CXCR4 and CCL27–CCR10 ratios were higher in thin than in thick primary melanomas, and all four chemokine-receptor ratios were higher in primary tumors than in melanoma metastases. CCL27–CCR10 and CXCL12–CXCR4 expression ratios in primary tumors were inversely associated with the development of distant metastases, and improved the predictive value of tumor thickness for distant metastasis, which is important since chemokine ligand-receptor ratios are not affected by the endogenous gene employed for normalizing mRNA expression. Both receptor and ligand immunolabeling were detected in neoplastic cells suggesting autocrine mechanisms. Our results support the concept that low CCL27/CCR10 and CXCL12/CXCR4 intratumoral mRNA ratios are associated with melanoma progression, and in combination with Breslow thickness, are the best predictive factors for the development of distant metastases in primary cutaneous melanoma.

Subcellular fractionation of actively growing protoplasts of Saccharomyces cerevisiae

Cell homogenates obtained from partially regenerated Saccharomyces cerevisiae protoplasts were fractionated by a procedure using a combination of continuous and discontinuous sucrose gradients, under experimental conditions that minimize possible artifacts due to centrifugation and resuspension. At least five different membranous organelle fractions (plasma membrane, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum-like structures and small-sized particulated structures) were isolated. Subcellular fractions were characterized by assaying established marker enzymes. Radioactive labelled [(U-3H]uracil) ribosomes were also used as a further characterization criterion of the rough endoplasmic reticulum. Comparative SDS-polyacrylamide gel electrophoresis of the protein constituents of the isolated membrane-bound organelles suggest that the polypeptide pattern could also be used as an additional marker for some of these structures. Finally, subcellular distribution of chitin synthase was determined using this fractionation procedure, and two partially zymogenic enzyme pools (one inside the cell associated to particles which sediments at high speed, and the second one associated to the plasma membrane) were found.

Candida albicans ABG1 gene is involved in endocytosis

The human fungal pathogen Candida albicans undergoes reversible morphogenetic transitions between yeast, hyphal and pseudohyphal forms. The fungal vacuole actively participates in differentiation processes and plays a key role supporting hyphal growth. The ABG1 gene of C. albicans encodes an essential protein located in the vacuolar membranes of both yeast and hyphae. Using fluorescence microscopy of a green fluorescent protein-tagged version of Abg1p, a fraction of the protein was detected in hyphal tips, not associated with vacuolar membranes. Live cell imaging of emerging germ tubes showed that Abg1p migrated to the polarized growth site and colocalized with endocytic vesicles. Phenotypic analysis of a methionine-regulated conditional mutant confirmed that Abg1p is involved in endocytosis.

Immunodetection of CD45 Epitopes on the Surface of Candida albicans Cells in Culture and Infected Human Tissues

Candida albicans is a leading cause of disseminated fungal infection in immunocompromised patients. Candida-host cell interactions are mediated at the cell surface. Since blood-group I epitopes have been detected on the surface of C albicans cells, we investigated whether CD45, the molecule that carries the I antigen on human lymphocytes, is present on the C albicans cell surface, in culture and in human tissue specimens of human candidiasis. By using monoclonal antibodies to CD45, CD45RO, and CD45RA, we found a strong immunoreactivity at the cell surface of blastoconidia bearing germ tubes but weak or no immunostaining of the germ tubes themselves. In human tissues, immunostaining of C albicans yeast cells was detected, whereas pseudohyphae were mostly negative. CD45 epitopes on the surface of C albicans might have a role in tissue invasion and dissemination of the fungus. On the other hand, its detection may disturb quantitative non-morphology-based determinations of lymphoid cell populations in infected tissues.