0000000000287838
AUTHOR
Salvador Mormeneo
Structural mannoproteins released by β-elimination fromCandida albicanscell walls
Abstract Mild alkaline solutions (β-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 β-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 ch…
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°C and 37°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- …
Monoclonal antibody 3H8: A useful tool in the diagnosis of candidiasis
In a previous series of experiments six mAbs were obtained against cell wall extracts of Candida albicans ATCC 26555. After several studies only one of them, designated 3H8, has been used to produce a commercial kit for the rapid diagnosis of candidiasis, Bichro-latex albicans (Fomouze Diagnostics). The present study involved the generation and characterization of this mAb as an immunoglobulin G1 which recognizes mannoproteins of high molecular mass present in the C. albicans cell wall. ELISA assays showed that the presence of the epitope recognized by mAb 3H8 was similar in both yeast and mycelial cell walls of C. albicans, in contrast to the epitope for mAb 1B12, which is mainly expressed…
Preparation of Anti-protein and Anti-mannan Antisera against Fungal Cell Wall by Affinity Chromatography
Abstract Iranzo, M., Marcilla, A., Elorza, M. V., Mormeneo, S., and Sentandreu, R. 1994. Preparation of anti-protein and anti-mannan antisera against fungal cell wall by affinity chromatography. Experimental Mycology 18, 159-167. A novel and easy chromatographic method has been developed for the isolation of anti-protein and anti-mannan antisera from a population of polyclonal antibodies obtained against Candida albicans and Yarrowia lipolytica cell wall mannoproteins. The technique is based on the immobilization of mannan (to be used as immunoadsorbent) by Affi-Prep H z resin after the oxidation of neighboring hydroxyl groups of the polysaccharide with sodium periodate. For Y. lipolytica p…
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 bond…
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 …
A rapid spectrofluorimetric method for the determination of the degree of synchronism inSaccharomyces cerevisiae
A fluorescence technique that allows direct measurement of DNA and synchrony levels inSaccharomyces cerevisiae cell cultures is reported. The spectrofluorimetric estimation of DNA with 4,6-diamidino-2-phenylindole (AT-dye) does not require prior extraction, is highly stable, and requires small quantities of cells.
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 signle 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…
Possible Roles of Mannoproteins in the Construction of Candida Albicans Cell Wall
The shape of Candida albicans cells depends on their cell walls and some of their mannoproteins may act as modulators of the final molecular architecture. If that were the case, the wall mannoproteins might form part of what could be called a “morphogenetic code”.
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 inc…
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…
Initial steps of wall protoplast regeneration in Candida albicans
Summary Cell wall regeneration of individual Candida albicans yeast and mycelial protoplasts was studied with confocal and electron microscopy using polyclonal antibodies and leetins. 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 β(1,3)- and β(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 foun…
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 o…
Biogenesis of the Fungal Cell Wall
Cell walls play essential roles in growth, development, and in interactions of fungi with the environment and with other cells. Besides its primary protective role in shielding the cell against osmotic, chemical, and biological harm, the wall is involved in many other functions including morphogenesis, and some activities that may be denominated as “social”, such as morphological responses, antigenic expression, adhesion, and cell-cell interaction (Peberdy 1990; Ruiz-Herrera 1992; Sentandreu et al. 1991). There are many data supporting the idea that temporal and spatial regulation of wall polymer synthesis and assembly are critical for the properties of the walls, which thus do not exclusiv…
Relationships Between Dimorphism, Cell Wall Structure, and Surface Activities in Candida albicans
Most cells are covered with a complex network of interacting molecules that form the extracellular matrix. These molecules (proteins and polysaccharides) are secreted locally and interact among themselves to form an organized structure outside the cell plasma membrane. In unicellular eukaryotic organisms and plant cells, this structure is reinforced to withstand osmotic changes in the external environment, giving rise to the so-called cell wall.
Characteristics of rice straw and sewage sludge as composting materials in Valencia (Spain).
This work supports the idea that composting can be useful for minimizing the rice straw and sewage sludge environmental impact. Several physical, chemical and microbiological properties of these raw materials were analyzed. The characteristics of the rice straw were complementary to those of the sewage sludge for the application of composting. The C/N ratios suitable for a rapid increased in microbial activity were the lowest (17-24). A temperature of 62 degrees C during 48 h removed pathogenic microorganisms from rice straw and sewage sludge mixture. The results obtained in the present work suggested that these materials could be use in the composting process.
Temporal aspects of the O-glycosylation of Saccharomyces cerevisiae mannoproteins
Abstract Cleavage of the O-glycosyl bonds of Saccharomyces cerevisiae cell wall mannoproteins by β-elimination resulted in the release of about 8% of the carbohydrate in the form of mannose and other low molecular weight oligomannosaccharides (mannose to mannopentaose), leaving 92% mannose still covalently linked to the peptide, and suggesting that this alkali-resistant fraction was N-glycosidically linked. At the non-permissive temperature, S. cerevisiae sec mutants accumulated in the cytoplasm mannoproteins with different degrees of O- and N-glycosylation. The glycoproteins of mutant sec 20-1 contained 60% of the carbohydrate linked by N-bonds, the remainder being O-glycosidically linked.…
The use of trypsin to solubilize wall proteins from Candida albicans led to the identification of chitinase 2 as an enzyme covalently linked to the yeast wall structure
The use of trypsin to break proteins covalently linked to the yeast walls of Candida albicans released approx. 50% of the proteins, but also glucose and N-acetylglucosamine. Analysis by affinity chromatography indicated that glucose and/or N-acetylglucosamine formed part of the same supramolecular complexes with mannoproteins. These complexes would represent a new type of cell wall structuration in which beta-1,6 glucan and chitin are linked to proteins. An internal peptide from a 50-kDa protein released by trypsin was sequenced, showing 100% identity with chitinase 2 protein and 92% with chitinase 3. The electrophoretic mobility of the chitinase 2 protein was changed by treatment with Endo…
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%).
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).
Analysis of pharmaceutical biodegradation of WWTP sludge using composting and identification of certain microorganisms involved in the process.
Pharmaceuticals (PhCs) are organic contaminants that have been detected in wastewater, surface water, and soils throughout the world. The presence of 10 commonly used PhCs in Spain (azithromycin, benzylpenicillin, citalopram, fluconazole, fluoxetine, ibuprofen, irbesartan, olanzapine, telmisartan, and venlafaxine) was analysed at four wastewater treatment plants, and the changes in their concentrations during treatment were assessed. Although certain some PhCs were degraded in the treated water, their presence in sewage sludge increased in all cases. The sewage sludge was composted using rice straw to degrade the PhCs, and the composting efficiency was modified by changes in the relative C/…
Determination of enzymatic activities using a miniaturized system as a rapid method to assess soil quality
Summary Soil quality determination requires the analysis of a number of soil attributes using different approaches. In recent years, one of the most promising approaches has been the determination of enzymatic activities. Generally, only a few enzymes have been analysed and related to other soil properties such as total carbon, nitrogen content or microbial biomass carbon. The aim of this work was to investigate the possible use of the API ZYM strip, a semi-quantitative miniaturized system that determines 19 enzymatic activities, to study soil quality. To this end, we tested the system in different soil types, including albic Arenosols, mollic Leptosols, rendzic Leptosols, haplic Leptosols …
Transglutaminase activity is involved in Saccharomyces cerevisiae wall construction
Transglutaminase activity, which forms the interpeptidic cross-link N(epsilon)-(gamma-glutamyl)-lysine, was demonstrated in cell-free extracts of Saccharomyces cerevisiae by incorporation of [(14)C]lysine into an exogenous acceptor, N,N'-dimethylcasein. Higher levels of the activity were present in the cell wall, which also contained endogenous acceptors. The enzyme activity in the wall was inhibited by cystamine, a known inhibitor of transglutaminase, and by EDTA, indicating a cation-dependent activity. After the endogenous wall acceptors were labelled radioactively by transglutaminase, extraction with SDS solubilized about 50% of the total radioactivity, while Zymolyase and chitinase each…
The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
Candida albicans is an opportunistic fungus that is part of the normal microflora commonly found in the human digestive tract and the normal mucosa or skin of healthy individuals. However, in immunocompromised individuals, it becomes a serious health concern and a threat to their lives and is ranked as the leading fungal infection in humans worldwide. As existing treatments for this infection are non-specific or under threat of developing resistance, there is a dire necessity to find new targets for designing specific drugs to defeat this fungus. Some authors reported the presence of the transglutaminase activity in Candida and Saccharomyces, but its identity remains unknown. We report here…
Secretion, interaction and assembly of two O-glycosylated cell wall antigens from Candida albicans.
The mechanisms of incorporation of two antigens have been determined using a monoclonal antibody (3A10) raised against the material released from the mycelial cell wall by zymolyase digestion and retained on a concanavalin A column. One of the hybridomas secreted an IgG that reacted with two bands in Western blots. Indirect immunofluorescence showed that the antigens were located on the surfaces of mycelial cells, but within the cell walls of yeasts. These antigens were detected in a membrane preparation, in the SDS-soluble material and in the material released by a 1,3-beta-glucanase and chitinase from the cell walls of yeast and mycelial cells. In the latter three samples, an additional h…
Wall formation by Candida albicans yeast cells: synthesis, secretion and incorporation of two types of mannoproteins.
SUMMARY: The mannoprotein components solubilized from the walls of Candida albicans blastoconidia following degradation of the glucan network with β-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 incuba…
Isolation and characterization of anavirulent Candida albicansyeast 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…