Manuel Casanova

Changes in the cell wall glycoprotein composition of Candida albicans associated to the inhibition of germ tube formation by EDTA

Hyphal development in Candida albicans was blocked by EDTA. This effect was not due to a general growth inhibition since the chelator did not affect protein and DNA synthesis. Recovery of mycelial growth was observed when EDTA-grown cells were incubated at 37°C in EDTA-free medium. High-molecular-weight mannoproteins (HMWM) that are mycelium-specific wall components, and particularly a 260-kDa species (HMWM-260), were absent in the wall of cells grown under germination conditions in the presence of EDTA. Synthesis of the HMWM-260 species was not inhibited but its incorporation (secretion) into the wall structure seemed to be blocked in EDTA-treated cells.

Wall mannoproteins in cells from colonial phenotypic variants of Candida albicans

Candida albicans ATCC 26555 switched at high frequency (10(-1) to 10(-3)) between several phenotypes identified by colony morphology on a defined mineral amino-acid-containing agar medium supplemented with arginine and zinc (LAZ medium). When cells taken from colonies exhibiting distinct morphologies were plated directly onto LAZ agar, spontaneous conversion to all the variant phenotypes occurred at combined frequencies of 2.1 x 10(-1) to 9.5 x 10(-3). However, when cells taken from the different colonial phenotypes were plated directly onto an undefined medium (yeast extract/peptone/dextrose; YPD medium), or first incubated in liquid YPD medium and then cloned on YPD agar, all colonies observed exhibited the same phenotype (smooth-white). When cells from the smooth-white colonies were plated as clones on LAZ agar, the original switch phenotype reappeared. These results suggest that environmental conditions such as the growth medium (and possibly the temperature) influence switching by suppressing phenotype expression, but have no effect on genotype. The variant colony morphologies also appeared to be associated with differences in the relative proportions of yeast and mycelial cells. Zymolyase digests of wall preparations obtained from cells belonging to different colonial phenotypes were analysed by SDS-PAGE. After blotting to nitrocellulose paper, the mannoproteins were stained with Concanavalin A, with a polyclonal antiserum enriched in antibodies against mycelium-specific wall components, and with a monoclonal antibody raised against a high-molecular-mass mannoprotein band (260 kDa) specific to the walls of mycelial cells. The results suggest that phenotypic switching might be associated with changes in the degree of glycosylation in high-molecular-mass mannoproteins, or in the way these mannoproteins are bound to other cell wall components.

Antigenic cell wall mannoproteins in Candida albicans isolates and in other Candida species

Polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs), raised against mannoprotein components from Candida albicans ATCC 26555 (serotype A) blastoconidia and mycelial cell walls, were used to investigate antigenic similarities among wall mannoproteins from other C. albicans serotype A and B strains, and from C. tropicalis and C. guilliermondii. Radioactively labelled walls isolated from cells grown at either 28 degrees C or 37 degrees C were digested with a beta-glucanase complex (Zymolyase 20T) to release cell-wall-bound mannoproteins. Numerous molecular species with different electrophoretic mobilities were released from the various isolates. Differences appeared to be related to both the organism and the growth temperature. Among the major protein components solubilized were mannoproteins larger than 100 kDa (high molecular mass mannoproteins), heterogeneous in size in most cases. Antigenic homology was detected among the cell wall high molecular mass mannoproteins of the two C. albicans serotype A isolates, whereas significant qualitative and quantitative differences were detected between serotype A and serotype B cell-wall-bound antigenic profiles. Moreover, C. tropicalis and C. guilliermondii wall antigenic determinants were not recognized by the preparations of pAbs and mAbs raised against C. albicans walls. A mannoprotein with a molecular mass of 33-34 kDa was present in the enzymic wall digests of all the organisms studied. When probed with pAbs raised against the protein moiety of the 33 kDa cell wall mannoprotein of Saccharomyces cerevisiae, antigenic cross-reactivity was observed in all cases except C. tropicalis. There appear to be significant antigenic differences between the mannoproteins of different isolates of C. albicans, and between those of C. albicans and other Candida species.

Diagnosis of systemic candidiasis by enzyme immunoassay detection of specific antibodies to mycelial phase cell wall and cytoplasmic candidal antigens

Diagnosis of systemicCandida infections was attempted by the use of an enzyme-linked immunosorbent assay (EIA) to detect IgG antibodies towards cell wall-bound and cytoplasmic candidal antigens. Cell wall antigens were sequentially solubilized by treatment of germinated blastoconidia ofCandida albicans (ATCC 26555 strain) with β-mercaptoethanol (βME extract) and digestion with Zymolyase 20T, a β-glucanase preparation (Zymolyase extract). Protoplasts obtained after treatment with Zymolyase were osmotically lysed (cytoplasmic antigens). Sera were obtained from patients with systemic (n=28) and superficial (n=46) candidiasis. Control sera were obtained from normal healthy individuals (n=31) and from hospitalized patients at low (n=36) and at high (n=13) risk of developing systemic candidiasis yet showing no symptoms of candidal infection. Detection of antibodies in crude sera samples by EIA using all of these antigenic extracts was highly specific (98–100 %), but sensitivity of the method was low (3.5–17.8 %). However, adsorption of sera with latex microspheres coated with purifiedCandida mannan in order to selectively remove antimannan antibodies prior to EIA improved the diagnostic efficiency of this test. Improvement was particularly noticeable when the βME extract was used as antigenic preparation, yielding a sensitivity of 89.2 % and a specificity of 98.6 %.

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.

Cell wall protein and glycoprotein constituents of Aspergillus fumigatus that bind to polystyrene may be responsible for the cell surface hydrophobicity of the mycelium

Cell surface hydrophobicity (CSH) of Aspergillus fumigatus grown both in complex medium (yeast extract/peptone/dextrose; YPD) and minimal (Vogels N) medium was monitored by assessing attachment of polystyrene microspheres to the cell surface. It was found that mature mycelium was hydrophobic. Treatment of intact mycelium with beta-mercaptoethanol (beta ME) abolished binding of the microspheres to hyphal elements, and coating of the microspheres with beta ME extracts from mycelium inhibited their attachment to intact mycelial cells. A. fumigatus mycelium was tagged in vivo with biotin and treated with beta ME. The beta ME extracts were analysed by SDS-PAGE and Western blotting with both peroxidase-conjugated-ExtrAvidin and concanavalin A (ConA). This procedure allowed identification of cell wall surface proteins and glycoproteins. Rabbit polyclonal antisera were raised against beta ME extracts obtained from cells grown in YPD and Vogels N media. These antisera defined some major cell-wall-bound antigens. SDS-PAGE and Western blotting analysis of the cell wall material released by beta ME and adsorbed on polystyrene microspheres revealed about 19 protein species with apparent molecular masses ranging from 20 to 70 kDa, and two high-molecular-mass glycoproteins of 115 and 210 kDa. Treatment of cells grown in YPD, but not those grown in Vogels N medium, with beta ME released a 55 kDa polypeptide able to adsorb to polystyrene microspheres that was detectable with the antisera. The ability to bind to polystyrene particles exhibited by several protein and glycoprotein species released by beta ME treatment suggested that these cell wall moieties possess exposed hydrophobic domains that could be responsible for the CSH of mycelium.

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 polydispersed 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 33 kDa 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.

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates of Candida albicans.

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.

Different Molecular Forms of Invertase in the slime Variant of Neuospora crassa: Comparison with the Wild-type Strain

SUMMARY: Invertase synthesis, regulation and secretion in the wall-less slime variant of Neurospora crassa was studied. Unlike the wild-type, synthesis of the enzyme was not repressed by glucose. This effect was not related to the os mutation harboured by the slime strain, nor to the phenotypic absence of a cell wall. Three molecular forms of extracellular invertase, which varied in size, were detected in the slime strain. These forms were interconvertible, with the equilibrium in favour of the larger form. Polypeptide analysis of the three separated forms revealed that all contained the same glycoprotein with an M r of 97000. This was completely deglycosylated by treatment with endo-β-N-acetylglucosaminidase H (Endo H) to a polypeptide with an M r of 72000. It was concluded that the three interconvertible forms correspond to the monomeric, dimeric and tetrameric states of the enzyme. Three similar forms of invertase, albeit of slightly different electrophoretic mobility, were found in cell-free extracts, cell walls and spent culture medium of the wild-type strain. After Endo H treatment, analysis showed that these forms contained a polypeptide that was equally reactive against anti-Saccharomyces cerevisiae antibodies, and had the same M r, as the polypeptide produced by the slime strain.