Avaliaçäo in vitro da fagocitose de Candida Albicans, sorotipos A e B, por macrófagos peritoneais ativados de camundongos isogênicos / An in vitro evaluation of Candida Albicans (serotypes A and B) phagocytosis, by activated peritoneal macrophages of isogenic mice

Tendo em vista a indefinida literatura em relaçäo aos macrófagos e à fagocitose de fungos e os resultados conflitantes frente a diversas metodologias empregadas, propusemo-nos, após o estabelecimento de um modelo experimental, estudar a fagocitose "in vitro" de Candida Albicans. Utilizamos para este estudo macrófagos peritoneais ativados, obtidos de camundongos isogênicos da linhagem A/Sn e duas cepas de Candida Albicans de diferentes procedências e sorotipos: ICB-12 sorotipo A e ICB-156 sorotipo B. Avaliamos a fagocitose pela contagem de macrófagos contendo em seu interior células vivas e/ou mortas, determinando-se o índice de fagocitose pela multiplicaçäo da porcentagem de macrófagos que fagocitaram e do número médio de leveduras por macrófagos. Valemo-nos de corantes vitais como vermelho neutro e fluorescentes (diacetato de fluoresceína e brometo de etídio), examinando o material à luz da microscopia de fase e fluorescência. Pudemos observar comportamento diferente dos macrófagos em relaçäo às duas cepas de C. Albicans estudadas, demonstrando porcentagem e índice maior de fagocitose dos macrófagos frente à cepa de C. Albicans ICB-12 sorotipo A quando comparada à cepa de C. Albicans sorotipo B, embora o número médio de leveduras por macrófagos fosse semelhante para ambas as cepas. Procurou-se, assim, proporcionar uma visäo sobre o destino de C. Albicans frente ao macrófago, devido aos vários aspectos ainda hoje näo totalmente definidos

Characterization of cell wall proteins of yeast and hydrophobic mycelial cells of Candida albicans.

Cell surface hydrophobicity (CSH) of blastoconidia and blastoconidia bearing germ tubes of Candida albicans ATCC 26555 was monitored by assessing attachment of polystyrene microspheres to the cell surface, and we found that mature hyphae were significantly hydrophobic. Treatment of intact cells with low concentrations of beta-glucanase (Zymolyase 20T) or proteases abolished or significantly reduced attachment of latex beads to hyphae. This effect paralleled an obvious reduction in CSH of the entire cell population, as measured by an aqueous-hydrocarbon biphasic partitioning assay. Analysis of the cell wall material released by Zymolyase and adsorbed on polystyrene microspheres indicated that germ tube-specific cell wall proteins and mannoproteins with apparent molecular masses of 20 to 67 kDa may be responsible for the hydrophobicity of hyphae. Zymolyase released from blastoconidia cell walls a different set of proteins and mannoproteins that were able to adsorb to polystyrene microbeads. Such molecular species might in turn be responsible for the CSH exhibited by blastoconidium populations as determined by the biphasic partitioning assay, although these probably hydrophobic components can be masked on the surface of blastoconidia, as the latter had no or very few latex microspheres attached to their surfaces. Treatment of cells of both C. albicans morphologies with 2-mercaptoethanol released qualitatively distinct species of polystyrene-adsorbed proteins and mannoproteins from yeast and mycelial cells. These observations suggested that hydrophobic proteins and mannoproteins that could be associated with CSH are bound to the cell wall structure through diverse types of linkages.

Candida albicans and three other Candida species contain an elongation factor structurally and functionally analogous to elongation factor 3.

A cell-free poly(U)-dependent translation elongation system from Candida albicans is ATP-dependent due to the presence of an elongation factor 3 (EF3)-like activity. Saccharomyces cerevisiae ribosomes added to a C. albicans postribosomal supernatant (PRS) supported poly(U)-dependent elongation, suggesting that the C. albicans lysate contained a soluble translation factor functionally analogous to the S. cerevisiae translation factor EF-3. The presence of EF-3 in C. albicans was confirmed by Western blotting using an antibody raised against S. cerevisiae EF-3. This antibody was also used to screen a selection of Candida species, all of which possessed EF-3 with molecular mass in the range of 110-130 kDa.

Cell wall glycoproteins of Candida albicans as released by different methods.

Different methods of extraction frequently used in other studies were used to release glycoproteins from both intact cells and isolated cell walls of yeast and hyphal forms of Candida albicans. Extracts were obtained from whole cells by treatment (i) with 2-mercaptoethanol (beta ME) at pH 8.6 and 37 C degrees and (ii) with zymolyase after treatment with beta ME. Extracts were obtained from isolated and washed cell walls (i) by boiling with beta ME and sodium dodecyl sulphate (SDS), (ii) by boiling with SDS and (iii) by treatment with zymolyase after SDS. The extracts were separated by SDS-polyacrylamide gel electrophoresis and analysed by Western blotting with four reagents. Analysis with concanavalin A (ConA) revealed different glycoprotein populations depending on the treatment. Three possible germ-tube-specific constituents were observed; and 80 kDa component released by beta ME from both intact cells and cell walls, and 47 kDa and 43 kDa moieties released by zymolyase only from intact cells. MAb 4C12, specific for the protein portion of a large germ tube constituent, recognized polydisperse material which just entered the gel in beta ME extracts and in the region extending up from 200 kDa to near the top of the gel in zymolyase extracts. MAb 24.17, specific for a carbohydrate determinant of yeast phase cells, reacted with disperse material in the region from the top of the gel to one-third to two-thirds the distance to the 220 kDa mass marker. Antiserum specific for the serotype A determinant of mannan reacted with large disperse component(s) migrating in the region from the top of the gel to about two-thirds the distance to the 220 kDa mass marker and with a 180 kDa component. The components recognized by MAb 4C12, but not those recognised by MAb 24.17 and serotype A antiserum, were effected by treatment with endo-beta-N-acetylglucosamidase H. The various analyses revealed that the method of extraction affected the composition and size of the constituents recognized by the reagents.

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.

Evidence for expression of the C3d receptor of Candida albicans in vitro and in vivo obtained by immunofluorescence and immunoelectron microscopy.

The complement conversion product C3d binds to a receptor on the cell surface of Candida albicans. While the function of this receptor is still uncertain, we investigated whether it is expressed during a murine infection. Rabbit antiserum raised against purified receptor was used in conjunction with immunofluorescence microscopy and immunocolloidal gold electron microscopy to examine kidney tissue and peritoneal lavages from infected mice for receptor expression by C. albicans in vivo. Specificity of the antiserum was indicated by reactivity with purified receptor (55 to 60 kDa) and with a protein of similar molecular mass from whole hyphal extracts in Western blots (immunoblots). In vitro analysis by immunofluorescence microscopy showed that the antiserum reacted with both yeast and pseudohyphal forms of the organism, but reactivity was strongest with pseudohyphae. Immunocolloidal gold electron microscopy of fungal cells from peritoneal lavages revealed intense staining of mother cells of germinative forms, germ tubes, and pseudohyphae. Staining of the mother cells was heaviest at the innermost layers of the cell wall but only scant on the cell surface. In contrast, staining was observed throughout the cell walls of germ tubes and pseudohyphae. In kidney, expression of the C3d receptor was found primarily on the cell walls of hyphae and pseudohyphae, although some staining was observed in the cytoplasm. These data support that the C3d receptor of C. albicans is expressed in vivo.

Phosphate-containing proteins and glycoproteins of the cell wall of Candida albicans.

The distribution of phosphate, carbohydrate, and protein in the cell wall components extracted from intact yeast cells of Candida albicans by beta-mercaptoethanol (beta ME) at pH 8.6 was examined by analysis of the material separated by DEAE-cellulose chromatography. All protein peaks did not coincide with peaks of both carbohydrate and phosphate. Subsequent analysis was performed on material obtained from yeast cells and germ tubes which were grown in medium containing [32P]phosphate. Two extracts were obtained by treating cells with beta ME or with zymolyase following beta ME. The extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. beta ME-extracted material contained high-molecular-mass (HMM), greater than or equal to 200 kDa, polydisperse material and a major and minor band of 19 to 20 kDa, Zymolyase extracts contained (i) three components of less than or equal to 40 kDa, one of which may correspond to the major beta ME band; (ii) four bands within the HMM region which may correspond to previously reported bands; and (iii) one band of 100 to 120 kDa. After longer exposures, additional midrange bands were detected in the zymolyase extract. In extracts treated with endo-beta-N-acetylglucosaminidase H, the HMM polydisperse material increased in mobility although retaining sufficient radiolabel for detection. Western immunoblot analysis of extracts with germ tube-specific antiserum and a germ tube-specific monoclonal antibody and concanavalin A showed that not all components contained detectable phosphate, not all glycoproteins contained detectable phosphate, and at least one 19- to 20-kDa protein may be phosphorylated in the absence of carbohydrate.

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.

Partial biochemical characterization of cell surface hydrophobicity and hydrophilicity of Candida albicans.

Hydrophobic yeast cells of Candida albicans are more virulent than hydrophilic yeast cells in mice. Results of experiments performed in vitro suggest that surface hydrophobicity contributes to virulence in multiple ways. Before definitive studies in vivo concerning the contribution of fungal surface hydrophobicity to pathogenesis can be performed, biochemical, physiological, and immunochemical characterization of the macromolecules responsible for surface hydrophobicity must be accomplished. This report describes our initial progress toward this goal. When hydrophobic and hydrophilic yeast cells of C. albicans were exposed to various enzymes, only proteases caused any change in surface hydrophobicity. Hydrophobic cell surfaces were sensitive to trypsin, chymotrypsin, pronase E, and pepsin. This indicates that surface hydrophobicity is due to protein. Papain, however, had no significant effect. The hydrophobicity of hydrophilic cells was altered only by papain. The proteins responsible for surface hydrophobicity could be removed by exposure to lyticase, a beta 1-3 glucanase, for 30 to 60 min. When 60-min lyticase digests of hydrophobic and hydrophilic cell walls were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 12.5% resolving gel, each protein population contained a single unique protein that was not evident in the other protein population. However, when the cell wall surface proteins of hydrophobic and hydrophilic cells were first labeled with 125I and then removed by lyticase and analyzed by SDS-PAGE, at least four low-molecular-mass (less than 65 kilodaltons) proteins associated with hydrophobic cells were either absent or much less abundant in the hydrophilic cell digests. This result was seen for both C. albicans strains that we tested. When late-exponential-phase hydrophilic cells were treated with tunicamycin, high levels of surface hydrophobicity were obtained by stationary phase. These results indicate that the surface hydrophobicity of C. albicans reflects changes in external surface protein exposure and that protein mannosylation may influence exposure of hydrophobic surface proteins.

A comparison of the sterol content of multiple isolates of the Candida albicans Darlington strain with other clinically azole-sensitive and -resistant strains.

The mechanism of azole resistance of Candida albicans NCPF 3310 (the deposited culture of the Darlington strain) has been investigated but never fully explained. Seven isolates of this strain, from various sources, were examined by gas chromatography-mass spectrometry to detect changes in the sterol composition following passage through many laboratories over several years. Five of the seven, including one recently isolated from the patient, were found to be similar to each other in sterol content, containing large amounts of fecosterol. Of the remaining two, one was thought to be a sensitive variant, both produced only small quantities of fecosterol and resembled the normal clinical strains and other azole-resistant strains in sterol content. The sterol composition of the Darlington strain was unique and apparently stable to prolonged in vitro experimentation and passage through the patient.

Mannan composition of the hyphal form of two relatively avirulent mutants of Candida albicans.

We have previously reported the characteristics of mannans isolated from the yeast forms of two relatively avirulent Candida albicans strains, designated 4918-2 and 4918-10. Investigations have been expanded to include an analysis of mannans from the hyphal form of these strains as well as from the hyphal form of the parental strain, 4918. After extraction, mannans were further purified by high-pressure liquid chromatography on a Bio-gel TSK DEAE-5-PW column. Subsequent to either mild acid hydrolysis, alkali hydrolysis, or acetylation followed by acetolysis, the resulting products were fractionated by high-pressure liquid chromatography on an Aminex HPX-42A column. The results of acid hydrolysis showed only minor quantitative differences in the products released from each strain, with mannose constituting the vast majority of product liberated. The profiles of mannooligosaccharides obtained from either alkali hydrolysis or acetolysis for strain 4918-2 showed distinct quantitative differences compared with profiles of the other two strains. Finally, a general characteristic noted is a decrease in the average chain length of mannooligosaccharides in hyphal mannans compared with the yeast counterpart.

Lipids of Candida albicans: subcellular distribution and biosynthesis.

Lipids constituted around 5% of the dry weight in Candida albicans 3153, while sterols and phospholipids accounted for 1.2% and 1.1% respectively. Phospholipids were mainly localized in the microsomal fraction; phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were the major phospholipids. Incorporation studies with [14C]acetate and [32P]orthophosphoric acid demonstrated that PS was synthesized at the highest rate followed by PC, PE and PI. There was little difference in either the content of the rate of biosynthesis of PC, PE and PI. Incorporation of labelled serine, ethanolamine and choline revealed serine to be a precursor for PC, PE and PS, ethanolamine for PC and PE, and choline for PC biosynthesis only.

Structural study of cell wall phosphomannan of Candida albicans NIH B-792 (serotype B) strain, with special reference to 1H and 13C NMR analyses of acid-labile oligomannosyl residues.

Chemical structures of manno-oligosaccharides, from biose to heptaose, released from the phosphomannan of Candida albicans NIH B-792 strain (serotype B) by mild acid hydrolysis were investigated. The results of 1H NMR, 13C NMR, and fast atom bombardment mass spectrometry analyses confirmed that these manno-oligosaccharides belong to a homologous beta-1,2-linked series. Although chemical shifts of 1H NMR patterns of these oligosaccharides were considerably too complicated to be assigned, their 13C NMR patterns were sufficiently simple to be interpreted, exhibiting a regular increase of downfield shift of ppm values of the C-1 atom from each mannopyranose residue in proportion to their molecular weights. In order to determine the whole chemical structure of the parent phosphomannan, the acid-stable domain was subjected to acetolysis and then enzymolysis with the Arthrobacter GJM-1 alpha-mannosidase and the resultant manno-oligosaccharides were investigated for their chemical structures by 1H NMR spectroscopy. The results of a precipitin-inhibition test using the beta-1,2-linked manno-oligosaccharides, from biose to hexaose, in comparison with the corresponding isomers containing alpha-1,2 linkage with small amounts of alpha-1,3 linkage, indicated that the haptens possessing the former linkage exhibited much higher inhibitory effects than the corresponding isomers containing the latter linkages did. Based on the present findings, a chemical structure of the phosphomannan of this C. albicans strain was proposed.

Purification and characterization of the extracellular C3d-binding protein of Candida albicans.

A C3d-binding glycoprotein was purified from the culture filtrate of Candida albicans by preparative isoelectric focusing. The protein possessed a pI of 3.9 to 4.1 and could inhibit rosetting of EAC3d (sheep erythrocytes conjugated to C3d) by pseudohyphae of C. albicans. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol, the protein migrated as a doublet with apparent molecular masses of 55 and 60 kilodaltons (kDa) and as a 50-kDa band in nonreducing gels. These results were observed with Aurodye stain for proteins. Western immunoblot, and concanavalin A stain, which indicates that both bands contain carbohydrate as well as antigenic determinants. The treatment of purified glycoprotein with endoglycosidase F but not endoglycosidases H, N, and O resulted in a complete conversion of the doublet into a faster-migrating broad band with an apparent molecular mass of 45 kDa. When the amino acid analysis of the C3d-binding protein was compared with that of the CR2 from B lymphocytes, significant differences were observed. These data indicate that C. albicans secretes a C3d-binding protein during growth in vitro which appears to be different from the mammalian C3d receptor.

Variation in lipid and sterol contents in Candida albicans white and opaque phenotypes.

In the white-opaque transition, cells of Candida albicans strain WO-1 switch reversibly and at high frequency between phases which differ both in colony and cellular phenotype. The lipid and sterol contents of the two phases were compared. White cells were higher in lipid and sterol contents in both mid-exponential and stationary phase cultures. In mid-exponential phase cultures, the lipids of white cells accumulated substantial amounts of apolar compounds, including steryl esters, alkyl esters, triacylglycerols, fatty acids, free sterols and mono- and di-glycerides, while opaque cells accumulated nearly equal proportions of apolar and and polar compounds, mainly phosphatidylethanolamines and phosphatidylcholines. In stationary phase cultures, both white and opaque cells had slightly higher proportions of polar lipids. Major differences in the lipid composition between white and opaque cells involved the contents of free sterols and derivatives of sterols. White cells contained higher proportions of free sterols than opaque cells, while opaque cells contained more steryl glycosides and steryl esters (approximately 2.5 times higher). Comparison of the sterols of the white and opaque cells by UV, TLC and GLC showed that a qualitative as well as quantitative difference exists between the two phenotypes. Fatty acid analysis of white and opaque cells showed that C-16 and C-18 fatty acids are the most abundant in both phenotypes. White and opaque cells varied in their fatty acid composition. The former had higher proportions of palmitoleic (16:1) and stearic (18:0) but lower proportions of linoleic (18:2) fatty acids than opaque cells. Analysis of fatty acids of major lipid classes present in both forms showed that fatty acid pattern varied dramatically according to whether the class had been isolated from white or opaque cells. Our results suggest that the lipid composition (particularly sterol and polyunsaturated fatty acids) of the opaque phenotype resembles that of mycelial cultures. Opaque cells showed more resistance to amphotericin B, nystatin, 5-fluorocytosine (flucytosine) and miconazole nitrate than white cells.

Effects of terconazole and other azole antifungal agents on the sterol and carbohydrate composition of Candida albicans.

The effects of terconazole, a triazole antifungal, on the sterol and carbohydrate composition of Candida albicans was compared with that of three imidazoles: clotrimazole, miconazole, and butoconazole. Exposure of C. albicans to terconazole resulted in a profound depletion of ergosterol with a corresponding increase in lanosterol content versus control cells. Carbohydrate analysis revealed a significant (245%) increase in chitin and a minimal effect on glucan and mannan in terconazole-treated cells. Similar effects on sterol and carbohydrate composition were observed with clotrimazole and miconazole. Butoconazole had a similar effect on sterol composition but had no effect on carbohydrate composition. The decreased ergosterol and increased lanosterol content is consistent with 14 alpha-demethylase inhibition by terconazole and the other azoles. The increase in cell wall chitin is most likely due to deregulation of chitin synthesis secondary to ergosterol depletion in the cell membrane. Because both chitin and ergosterol are critical components of the fungal cell, perturbation of the production and localization of these components by terconazole is likely to contribute to the selective toxicity of this compound for C. albicans and other fungi.

Cell-constituent polyamines in Candida species and new biotyping of Candida albicans, Candida tropicalis and Candida parapsilosis strains.

Intracellular amines from three Candida species were extracted and chemically derivatized by a modification of the Seiler procedure. The results of qualitative determinations of 13 amines in 20 strains each of Candida albicans, Candida tropicalis and Candida parapsilosis are presented. This report is the first to describe the detection of amines other than the classical spermine, spermidine, putrescine and cadaverine in yeasts and fungi. Characteristic profiles due to the presence or absence of particular amines in the Candida species studied are demonstrated. Although these could not be used as strict differential markers at the species level, biotyping schemes based on amines are proposed to differentiate strains of C. albicans, C. tropicalis and C. parapsilosis.

Dynamic changes of the cell wall surface of Candida albicans associated with germination and adherence.

The distribution of mannoproteins at the cell wall surface of Candida albicans was analyzed during the process of germination in conditions favoring adherence of germ tubes to a plastic matrix. Three cytochemical methods allowing the detection of concanavalin A binding sites, anionic sites and the enzyme acid phosphatase, respectively were used. All three methods gave similar results, indicating a spatial and temporal reorganization of some cell wall mannoproteins: a strong labeling was observed on blastoconidia; in contrast, as soon as the emergence of germ tubes took place, these reactions decreased dramatically at the surface of mother cells, whereas the germ tube surface was strongly stained. Some new components with multiple biological activities were detected at the germ-tube surface. Indeed, among mannoproteins responsible for an enhanced adhesion to plastic surfaces, two components with molecular weights of 68 and 60 to 62 kDa were shown to interact with laminin, fibrinogen, and C3d. This study therefore indicates that germination, and then adherence of germ tubes, imply a degradation of surface mannoproteins, and a simultaneous presentation of new molecules which can interact with their nonbiological materials or host proteins.