Isolation and characterization of an avirulent Candida albicans yeast monomorphic mutant.

Mutagenesis of Candida albicans strain ATCC 26555 with N-methyl-nitro-N-nitrosoguanidine followed by plating on solid yeast nitrogen base-N-acetylglucosamine medium at 37 degrees C yielded colony morphology variants that were characterized as forming smooth colonies, in contrast to the rough colonies formed by the parental strain. One yeast monomorphic mutant, CAL4, was studied in detail. Strain CAL4 is defective in filamentous growth, unable to form hyphae or pseudohyphae in vivo and in vitro. These filamentous structures are not elicited by commonly used external stimuli such as serum. The mutant had no obvious alterations in its mannan, glucan or chitin content. The total quantity of non-covalently linked wall proteins was reduced in the mutant strain, but the electrophoretic pattern shown by these proteins was identical to that of proteins from the parental strain. CAL4 showed major differences from the parental strain in its formation of covalently linked wall proteins. An important aspect of these differences lay in the practical absence of proteins recognized by two monoclonal antibodies, 1B12 and 3H8, which are considered valuable tools in the diagnosis of candidiasis in part because they normally react strongly with all strains. The C. albicans mutant, blocked in yeast-mycelium transition, was avirulent in a mouse model, although it was able to grow in animal tissues.

Reaggregation and binding of cell wall proteins from Candida albicans to structural polysaccharides.

Urea or hot sodium dodecyl sulphate extracted a significant amount of the same proteins from the matrix of the cell wall of the yeast form and mycelial cells of Candida albicans. Gel filtration analysis of the urea-extracted proteins revealed that they occurred in the form of large complexes which were unaffected by up to 8 M urea. Among them, proteins en route to becoming covalently associated within the wall scaffold were identified by their reaction with specific antibodies. When urea was removed by dialysis, some of these proteins specifically reassociated into large aggregates which bound strongly with ConA, whereas others remained soluble in smaller associated products. The ability of some of these proteins to bind to the insoluble wall polysaccharides was also assessed. No self-assembling proteins were able to bind to glucans and/or chitin. Specificity of the binding to polysaccharides made of beta-bound glucosyl or N-acetylglucosaminyl residues was determined by the competitive effect of several disaccharides. Whereas laminaribiose and diacetylchitobiose were strong inhibitors of protein binding to both glucan and chitin, lactose, maltose and sucrose were ineffective.

Initial steps of wall protoplast regeneration in Candida albicans.

Cell wall regeneration of individual Candida albicans yeast and mycelial protoplasts was studied with confocal and electron microscopy using polyclonal antibodies and lectins. Quantitative measurements of the fluorescence emitted by individual protoplasts during the process of regeneration indicate that chitin is the first polymer to be laid down, whereas beta (1,3)- and beta (1,6)glucan are incorporated at a later stage. Mannoproteins were found on the surface of fresh protoplasts and those newly synthesized were then deposited with time. During the first steps of wall regeneration, the proteins that interacted covalently with chitin or glucan were different, but the same species were found linked to each polymer in yeast and mycelial regenerating forms. The aggregates formed by regenerating protoplasts were shown to be due to the chitin and mannoprotein network initially laid.

Study of supramolecular structures released from the cell wall of Candida albicans by ethylenediamine treatment.

Candida albicans cell wall components were analyzed by ethylenediamine (EDA) treatment. Based on their different solubility properties, the cell wall components produced three fractions (A, B, and C). Fractions B (EDA-soluble, water-insoluble) and C (EDA-insoluble) contained glucan, chitin, and protein in different proportions. After zymolyase (mainly a beta-glucanase complex) or chitinase treatment of fractions B and C, more polysaccharides and proteins were solubilized by a second EDA treatment, suggesting that the solubility of the polymers in EDA depends on the degree of polymer interactions. Western blot analysis using two monoclonal antibodies (1B12 and 4C12) revealed electrophoretic patterns that were similar in mycelial and yeast morphologies, except that in material obtained from mycelial walls, an additional band was detected with MAb 1B12. Fluorescence microscopy of cell wall fractions treated with FITC-labeled Con-A, Calcofluor white, and FITC-labeled agglutinin showed that glucan and mannoproteins are uniformly distributed in fractions B and C, while chitin is restricted to distinct patches. Transmission electron microscopy demonstrated that fraction C maintained the original shape of the cells, with an irregular thickness generally wider than the walls. When fraction C was treated with chitinase, the morphology was still present and was maintained by an external glucan layer, with an internal expanded fibrillar material covering the entire cellular lumen. Degradation of the glucan skeleton of fraction C with zymolyase resulted in the loss of the morphology.

Involvement of transglutaminase in the formation of covalent cross-links in the cell wall of Candida albicans.

Activity of the enzyme glutaminyl-peptide--glutamylyl-transferase (EC 2.3.2.13; transglutaminase), which forms the interpeptidic cross-link N epsilon-(gamma-glutamic)-lysine, was demonstrated in cell-free extracts obtained from both the yeast like and mycelial forms of Candida albicans. Higher levels of enzymatic activity were observed in the cell wall fraction, whereas the cytosol contained only trace amounts of activity. Cystamine, a highly specific inhibitor of the enzyme, was used to analyze a possible role of transglutaminase in the organization of the cell wall structure of the fungus. Cystamine delayed protoplast regeneration and inhibited the yeast-to-mycelium transition and the incorporation of proteins into the cell wall. The incorporation of covalently bound high-molecular-weight proteins into the wall was sensitive to cystamine. Proteic epitopes recognized by two monoclonal antibodies, one of which is specific for the mycelial walls of the fungus, were also sensitive to cystamine. These data suggest that transglutaminase may be involved in the formation of covalent bonds between different cell wall proteins during the final assembly of the mature cell wall.

Isolation and partial characterization of uronic acid-containing glycoproteins from Mucor rouxii.

Five different fractions containing uronic acids associated with protein were isolated from the cytoplasm of the filamentous form of Mucor rouxii. A single fraction was isolated from the cell wall by hot sodium dodecyl sulfate followed by ion exchange column chromatography. Two cytoplasmic entities (peaks I and II) were not adsorbed to DEAE Bio-Gel A. The molecular mass of peaks I to V ranged from 16.5 to 210 kDa. The protein-uronic acid ratios were different for each fraction. The cell wall fraction showed a molecular mass of 16.5 kDa, similar to that of peak II but with differences in chromatographic behavior and protein-uronic acid ratio. The possible role of these molecules as acceptors of sugar residues during polyuronide chain growth is discussed.

Specific immunohistochemical identification of Candida albicans in paraffin-embedded tissue with a new monoclonal antibody (1B12).

In invasive candidiasis, the identification of Candida organisms in tissue samples or in normally sterile fluids is essential for an accurate diagnosis. Species identification is an important clue for the source of infection and in epidemiological studies. In this article, the authors have tested the value of a new monoclonal antibody (1B12) to detect C albicans in culture by immunofluorescence, and in tissue samples by immunohistochemistry. MAb 1B12 was found to specifically recognize C albicans, does not cross-react with other Candida species or other structurally similar fungi, and is very sensitive and specific in paraffin-embedded tissue, having no reactivity in normal human tissues or necrotic areas. Therefore, MAb may be a valuable tool in the evaluation of fungal infections in paraffin-embedded tissue, particularly when Candida species identification is needed.

Structural mannoproteins released by beta-elimination from Candida albicans cell walls.

Mild alkaline solutions (beta-elimination), after removing the non-covalently bonded wall materials by hot SDS, released 13% and 26% of remaining wall proteins from mycelial and yeast cells of Candida albicans, respectively. When the beta-elimination was carried out after digestion of the walls with chitinase, four-fold more proteinaceous materials were released from mycelium and a similar amount in yeast walls. The solubilized materials were shown to be highly polydisperse, and endo-glycosidase H reduced their polydispersity and molecular masses, revealing different electrophoretic patterns in yeast and mycelial cell walls. The solubilized mycelial proteins carried N-glycosidic sugar chains and the epitopes recognized by two monoclonal antibodies were preserved, although showing a different behaviour in yeast walls. These results are consistent with the idea that significant amounts of intrinsic O-glycosylated mannoproteins are interconnected in the walls of C. albicans.

Structural organization of the components of the cell wall from Candida albicans.

The organization of the components of the cell wall from Candida albicans was studied by means of sequential treatment with hot SDS, anhydrous ethylenediamine (EDA) and lytic enzymes, followed by chemical and microscopic analyses of the different separated fractions. The EDA-insoluble fraction retained the original morphology of the wall, which was destroyed by beta-glucanase, but not by chitinase treatments. Staining with fluorescent lectins revealed distinct distributions of mannoproteins, glucans and chitin in the wall. Amino acid analysis of SDS-extracted walls, and the EDA-soluble and -resistant fractions gave similar results, with seven amino acids making up about 70% of the total protein weight. Treatment of the EDA-insoluble fraction with Zymolyase or chitinase released fragments of variable size whose susceptibility to these and other hydrolases suggests that they are made of glucan, chitin and mannan oligomers associated with proteins. Treatment of the Zymolyase-insoluble residue with chitinase released a series of low-molecular-mass oligomers made of neutral sugars, GlcNAc and amino acids, mainly lysine. It is suggested that they represent fragments of the core making up the scaffold of the cell wall of the fungus.

Incorporation of specific wall proteins during yeast and mycelial protoplast regeneration in Candida albicans.

The kinectics of incorporation of two precursor mannoproteins into the regenerating cell wall of Candida albicans protoplasts have been followed at 28 degrees C and 37 degrees C using two monoclonal antibodies specific for protein epitopes (MAb 1B12 and 4C12) as probes. Both molecules were secreted from the beginning of the regeneration process, and their incorporation was retarded significantly. Analysis of the secreted materials by Western immunoblotting with MAb 1B12 allowed the identification of two closely migrating bands at apparent Mr higher than 170 kDa and significant amounts of a highly polydisperse material of even greater molecular mass. Some of these mannoproteinaceous species carried both N- and O-glycosidically linked mannose residues, as deduced from their drop in apparent Mr when synthesized in the presence of tunicamycin and by their reactivity with Concanavalin A. Following secretion, the molecules reacting with MAb 1B12 were incorporated into the regenerating walls by covalent binding. Then, when the antigen molecules were solubilized from partially regenerated walls, their mobility differed when regeneration took place at 28 degrees C (blastoconidia) or 37 degrees C (mycelial cells).

Wall formation by Candida albicans yeast cells: synthesis, secretion and incorporation of two types of mannoproteins.

The mannoprotein components solubilized from the walls of Candida albicans blastoconidia following degradation of the glucan network with beta-glucanase (Zymolyase) have higher molecular masses than their probable precursors present in the supernatant of regenerating protoplasts. It therefore appears that the mannoproteins are released from the walls as part of supramolecular complexes. Immunological analysis using both polyclonal and monoclonal antibodies has demonstrated the probable relationship between molecules found in a mixed membrane preparation, those secreted by regenerating protoplasts, and those present in yeast cell walls. Some mannoproteins secreted by protoplasts incubated in the presence of tunicamycin had significantly increased mobility on SDS-PAGE, whereas others were not affected by the treatment. It is therefore possible that two types of mannoproteins are secreted by protoplasts: one carrying N-glycosylated chains (mannan) and one lacking them. All the proteins secreted in the presence of tunicamycin stained with Concanavalin A-peroxidase, demonstrating that they all, including the N-glycosylated ones, carried O-glycosylated sugar residues. Both classes of mannoproteins, secreted independently of each other, were found in the molecular complexes rendered soluble from the wall by Zymolyase digestion. Data obtained with a monoclonal antibody demonstrated the presence of a repeated epitope within one wall protein(s) detectable in a mixed membrane preparation and in the wall complexes released by Zymolyase.

Linkages between macromolecules in Candida albicans cell wall.

Wall mannoproteins can be divided into two major groups depending upon their degree of interaction with the structural network: one type interacts by non-covalent bonds while the second group seems covalently bound to other wall components (intrinsic or structural mannoproteins). Cytological and biochemical studies have shown that mannoproteins are distributed randomly throughout cell wall interacting with glucan, chitin and other mannoproteins. Experimental results obtained using regenerating protoplasts have shown that building of the wall occurs in two steps: during the first one the skeleton of chitin is formed retaining protein molecules by non-covalent bonds. The second steps take place by deposition of glucan molecules that allows incorporation of mannoproteins probably by covalent linkages. Using two monoclonal antibodies, one of them reacting with a mycelial specific antigen and a second one with an epitope found in yeast and mycelial cells, two families of proteins are detected, that are secreted independently. The antigens reacting with both monoclonal antibodies are solubilized from the walls of each morphologic structure forming part of supramolecular structures.

Candida albicans mycelial wall structure: supramolecular complexes released by zymolyase, chitinase and beta-mercaptoethanol.

Different techniques released from the wall of Candida albicans mycelial cells high molecular weight mannoprotein materials with different levels of complexity. SDS solubilized among others one protein of 180 kDa which reacted with a monoclonal antibody (MAb) specific of a O-glycosylated protein secreted by regenerating mycelial protoplasts [Elorza et al. (1989) Biochem Biophys Res Commun 162:1118-1125]. Zymolyase, chitinase and beta-mercaptoethanol, released different types of high molecular highly polydisperse mannoprotein materials (greater than 180 kDa) that also reacted with the same MAb. These materials had N-glycosidically linked sugar chains, in addition to the O-glycosidically bonded sugars, as their molecular masses were significantly reduced by Endo H digestion. Besides, the specific materials released by either zymolyase or chitinase seemed to be the same throughout the process of germ tube formation. Transmission electron microscopy of thin sections of cells and walls showed that mannoproteins and chitin are evenly distributed throughout the entire cell wall structure.

Evidence for the formation of covalent bonds between macromolecules in the domain of the wall of Candida albicans mycelial cells.

An O-glycosylated mannoprotein, after its incorporation into the wall, showed an increase in its molecular weight, due at least to its association with N-glycosidic sugar chain(s). This was shown by rendering the material soluble after partial degradation of the wall structure. At present it is unknown whether this phenomenon is due to an additional transglycosylation process or whether the partial degradation of the wall solubilizes a supramolecular structure formed between the original O-glycosylated protein which becomes linked either directly or indirectly through a protein to the N-sugar chain(s).

O-linked mannose composition of secreted invertase of Saccharomyces cerevisiae.

The secreted invertase (EC 3.2.1.26) of Saccharomyces cerevisiae is a glycoenzyme that contains N- and O-linked mannoses in 40/1 proportion. The small amount of mannose chains O-linked to invertase is distributed as follows: mannose (20%), mannobiose (50%), mannotriose (6%), mannotetraose (7%) and mannopentaose (17%).

Cellular location of asparaginase activity in Saccharomyces cerevisiae and regulation of this activity by nitrogen compounds.

Three asparaginase activities have been detected in Saccharomyces cerevisiae. One is found outside the permeability barrier; a second one is found inside and is soluble in the cell, and the third one is localized in a system of membrane particles. Synthesis of the membrane and external asparaginases require "de novo" synthesis of RNA and protein. The synthesis of exocellular asparaginase is inhibited by several nitrogen compounds (catabolite repression). This inhibition might take place at the transcriptional level. Moreover, this isoenzyme is reversibly inactivated by its natural substrates (catabolite inhibition by substrates). The half life of external asparaginase mRNA was calculated by two independent methods and values of 7.5 and 9.5 min were found.

Precise nucleotide sequence modifications with bidirectionally cleaving class-IIS excision linkers.

Bidirectionally cleaving blunt-ended DNA linkers have been constructed to generate defined nucleotide sequence modifications. The oligodeoxynucleotides (termed 'excision linkers'), contain two back-to-back recognition sites for class-IIS restriction endonucleases and provide a new instrument for modifying DNA primary structure. Following insertion of these linkers into host DNA, digestion with the cognate class-IIS enzyme results in a cleavage upstream and downstream from the adjoining enzyme recognition sites. Bidirectional cleavage efficiency can be improved by including spacer nucleotides between the two recognition sites. The number of nucleotides removed from or added to the host DNA depends upon the cleavage shift characteristic of the class-IIS enzyme, the design of the linker (including lateral spacer nucleotides to set the cleavage position), and the method used to make blunt ends from staggered ends following excision of the linker. BspMI linkers constructed in this study have been used to generate defined deletions in the ApR and TcR genes of pBR322. BsmI excision linkers are also described.

Molecular events associated with glucose repression of invertase in Saccharomyces cerevisiae.

When S. cerevisiae growing in the presence of glucose (repressive condition) was shifted to higher temperatures, invertase was secreted. This secretion required protein synthesis, but was independent of RNA formation (Mormeneo & Sentandreu 1982). In addition accumulation of invertasespecific messenger RNA occurred in the absence of protein synthesis but was expressed only after synthesis of protein. Invertase mRNA was continuously synthesized under repressive conditions and the levels of this mRNA were regulated by the presence of glucose. The hexose regulated the concentration of this mRNA at the level of transcription and/or by sensitization of this messenger RNA. The expression of the invertase mRNA present in the cells under repressive conditions was also regulated by glucose at the level of translation and/or secretion. As a result of these processes, under repressive conditions invertase is eliminated before secretion takes place.