Discriminant analysis of cellular fatty acids of Candida species, Torulopsis glabrata, and Cryptococcus neoformans determined by gas-liquid chromatography.

We used discriminant analysis of cellular fatty acid compositions determined by gas-liquid chromatography to differentiate yeastlike fungi (a total of 190 strains; including 37 Candida albicans strains, 21 Candida krusei strains, 13 Candida guilliermondii strains, 37 Candida tropicalis strains, 10 Candida pseudotropicalis strains, 24 Candida parapsilosis strains, 32 Torulopsis glabrata strains, and 16 Cryptococcus neoformans strains). Previous results with a standard strain of C. albicans indicated that reproducible fatty acid chromatograms can be obtained with cells grown in a medium of 2% Sabouraud glucose agar at 35 degrees C for between 48 and 72 h. These conditions were also maintained in cultures of the other organisms that we studied. The cellular fatty acid compositions of the organisms were determined quantitatively by gas-liquid chromatography and analyzed by discriminant analysis. The total correct identification expressed as relative peak percent was 95.8% (89.2% for C. albicans to 100% for C. krusei, C. guilliermondii, C. pseudotropicalis, T. glabrata, and C. neoformans). The total correct identification expressed as the common peak (palmitic acid) ratio was 94.7% (87.5% for C. parapsilosis to 100% for C. pseudotropicalis, T. glabrata, and C. neoformans). Both results suggest that cellular fatty acid compositions can be differentiated by this method.

Cell-wall glucans of Cryptococcus neoformans Cap 67.

Purified cell walls derived from Cryptococcus neofromans Cap 67, an acapsular mutant, consisted of 86% Glc and 7.3% GlcNAc. The integrity of the cell walls was disrupted in three successive extractions with 60% 4-methylmorpholine N-oxide (4-MMNO) at 120 degrees. Four 4-MMNO-soluble D-glucopyranans were isolated. Released within 0.5 h was water-insoluble Gi-1, followed by two water-soluble Gs fractions and water-insoluble Gi-2 over 17.5 h. A 4-MMNO-insoluble residue, containing 27% of GlcNAc, was also isolated. Gi-1 and Gi-2 were isolated as precipitates during dialysis of 4-MMNO extracts and were each reduced with NaBH4 to permit their investigation in alkaline solution. Gs-1 and Gs-2 were separated by ion-exchange chromatography of the water-soluble fractions. The structures of the D-glucopyranans were determined by 13C-n.m.r. spectroscopy and by g.l.c.-mass spectrometry of their per-O-methylated derivatives. Gi-1 was a (1----3)-alpha-D-glucopyranan (97%) with some (1----4)-D-glucosidic linkages (3%) and no chain-branching. Gs-1 and Gs-2 were (1----6)-beta-D-glucopyranans branched at O-3 (10-12%) with beta-D-Glcp-(1----3)-beta-D-Glcp side chains. Gs-2 may have approximately 2% more chain branching than Gs-1. Gi-2 was a D-glucopyranan with 80% of its structure like that of Gi-1, and 20% like that of Gs-1 and -2; the water-insolubility of Gi-2 suggests that these structures were covalently linked. Almost identical D-glucopyranans were obtained from aged cultures that had thickened walls (as observed by electron microscopy).

Diagnóstico de laboratorio de hongos del género cryptococcus, mediante un sistema convencional y uno computarizado / Laboratory diagnosis of fungi: of the genus Cryptococcus by conventional and computarized methods

Se estudiaron 6 cepas de hongos del género Cryptococcus aisladas de varios productos patológicos (líquido cefalorraquídeo, macerado de meninges y lesiones de piel) a las cuales se les aplicó un esquema de identificaciòn convencional y otro, utilizando técnicas de computaciòn que permitieron un determinación más rápida de las especies. Se analizan los posibles factores que se deben tener en cuenta en el diagnòstico de laboratorio de las cryptococcosis y se ofrecen los criterios de clasificaciòn

Type-specific polysaccharides of Cryptococcus neoformans. n.m.r.-spectral study of a glucuronomannan chemically derived from a Tremella mesenterica exopolysaccharide.

A glucuronomannan (GM) was derived by removal, through Smith degradation, of xylose from the native (3-O-acetylglucurono)xylomannan exopolysaccharide isolated from Tremella mesenterica. 13C-N.m.r. chemical shifts measured at various pD values were compared for p-nitrophenyl beta-D-glucopyranosiduronic acid (1) and two GMs (2 and 3) differing in GlcA content (Man:GlcA; 2, 10:1; and 3, 5:1). Also measured and compared were pKa values for 1 and 2. One-dimensional and two-dimensional (COSY and HETCOR) n.m.r. data allowed unambiguous assignments of pD-sensitive chemical shifts due to 2-O-beta-D-GlcpA substituents attached to a (1----3)-linked alpha-D-Manp backbone. The pKa and n.m.r. data indicated that the CO2H groups in either GM are independent of each other, and are similar in behavior to those of p-nitrophenyl beta-D-glucopyranosiduronic acid molecules. The n.m.r. data confirmed the previous, chemically deduced, structural role of GlcpA in the native polysaccharide from T. mesenterica, and indicated that significant pD-induced changes occur in the stabilities of the glycosidic orientations in the GM. Previous 13C-n.m.r. assignments for 2-O-beta-D-GlcpA in polysaccharides derived from Cryptococcus neoformans serotype A-variant were confirmed, except for the signal due to the anomeric carbon atom. This signal is now known to be pD-sensitive. In acidic solutions, it is coincident with the signal (104.5 p.p.m.) due to the anomeric carbon atoms of the unsubstituted alpha-D-Manp backbone residues. In basic solutions, the 2-O-beta-D-GlcpA anomeric carbon resonance is shifted upfield by approximately 0.2 p.p.m., and is observed as a separate signal.

[The structure of extracellular heteroglycans in various Cryptococcus species]. / Stroenie vnekletochnykh geteroglikanov nekotorykh vidov kriptokokkov.

Extracellular polysaccharides produced by some Cryptococcus species have been structurally investigated. These polymers have identical core structure, which was found to be alpha-1,3-mannan and different degrees of substitution of mannose in the core by xylose and glucuronic acid residues of side chains and different composition of side chains. Heteropolysaccharide from Cr. humicolus, the simplest one, has the same structure as the Cr. neoformans serotype D capsular polysaccharide. The Cr. skinneri polymer proved to be the most branched among Cryptococcus polysaccharides.

Carbohydrate patterns of Candida, Cryptococcus and Rhodotorula species.

Within the genus Candida three distinct groups are recognized on the basis of carbohydrate patterns of intact whole cell hydrolyzates. In the first, ascomycetous, group mannose is dominant, while rhamnose, fucose and xylose are absent; this is indicative of an affinity with endomycetous families. Among the basidiomycetous representatives, two groups can be recognized. One group is usually characterized by the presence of xylose and has a low mannose content. The pattern is typical for Cryptococcales and Tremellales (e.g., Cryptococcus, Trichosporon, Bullera and Tremella). The other basidiomycetous group is characterized by the presence of fucose and/or rhamnose with significant amounts of mannose. This pattern is characteristic for Sporobolomycetaceae.

Structure determination of Cryptococcus neoformans serotype A-variant glucuronoxylomannan by 13C-n.m.r. spectroscopy.

A series of polysaccharides was derived by physical and chemical methods from an antigenic, O-acetyl-containing, glucuronoxylomannan (GXM), isolated from the growth medium of Cryptococcus neoformans (CDC B2550) serotype A-variant having composition ratios of Man:Xyl:GlcA:OAc = 10:4:3:6. 13C-N.m.r. spectra of derivatives provided new structural evidence for GXM. Treatment of GXM with Li in ethylenediamine gave a xylomannan (XM, with Man:Xyl = 5:2). Smith degradation of XM gave a mannan (M). Ultrasonic treatment of GXM gave GXM-sonicated (GXMS). Treatment of GXM with 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide.HCl and then with NaBH4 gave reduced GXMS (RGXMS), or with aq. trifluoroacetic acid gave partially acid-hydrolyzed GXMS. Periodate oxidation of GXM and NaBH4 reduction of the product gave a polyalcohol-mannan (PM). Treatment of GXMS, RGXMS, and PM with NH4OH at pH 11 gave the respective O-deacetylated analogs. Comparison among the 13C-n.m.r. spectra of GXM, the various derivatives, and reference monosaccharides allowed the following conclusions: M is (1----3)-alpha-D-mannopyranan; XM consists of the M backbone with 91% of the Xyl on nonadjacent Man residues as 2-O-beta-D-Xylp substituents and with 9% as 4-O-D-Xylp substituents on other Man residues. GXM consists of the XM structure, but with non-D-xylosylated Man residues substituted with 2-O-beta-D-GlcpA substituents and with 6-O-acetyl groups distributed approximately equally on Man residues that have other substituents and those that have none. The molecular mechanics program MM2 was used to estimate the relative energies of anomeric orientations of the typical glycosidic linkage in M. The results suggest that 6'-OH----O-2 H-bonding is significant in the minimal-energy orientation of M, with phi = -36 degrees and psi = 51 degrees, and that two other glycosidic orientations may be important in the 2-O- or 6-O-substituted derivatives of M.

Fluorescence of Blastomyces and other fungi in Papanicolaou-stained pulmonary cytology preparations: comparison with light microscopy.

In this study, 152 Papanicolaou-stained pulmonary cytologic smears from 15 known cases of pulmonary fungal infection were randomly mixed with 194 control pulmonary smears. All slides were examined for fungi by three observers, first by light microscopy and then by fluorescent microscopy. The results of the light and fluorescent microscopy were compared. It was concluded that when both methods were used for fungal detection, the yield of positive results was higher.

Strain variation in composition and molecular size of the capsular polysaccharide of Cryptococcus neoformans serotype A.

The capsule of Cryptococcus neoformans is an important virulence factor. In this investigation capsular polysaccharides (CPSs) were isolated by ethanol precipitation from culture filtrates of C. neoformans serotype A strains 6, 15, 98, 110, and 145. Capsule sizes on India ink examination ranged from barely perceptible (strain 15) to greater than the diameter of the yeast cell (strain 6); the others were intermediate in size. On ion-exchange chromatography on DEAE-cellulose each CPS eluted at 0.2 M NaCl; CPS of strain 15 had two major peaks, designated III and IV. On gel-permeation chromatography CPSs of strains 6, 98, 110, and 145 eluted at the void volume of Sepharose CL-2B in the presence or 0.1 M EDTA, while the CPS of strain 15 eluted in two peaks. Sephacryl S-1000 resolved CPSs of all five strains in the following order, from largest to smallest molecular size: 145 greater than 110 greater than 98 greater than 6 much greater than 15. All five CPSs contained mannose, xylose, and glucuronic acid, while the carboxyl-reduced CPS of strain 110 also contained a large percentage of an inositol-like compound. The CPS of strain 110 contained approximately 30% uronic acid by weight, while the others had 15 to 20%. The composition of peak IV from the CPS of strain 15 resembled those of the other strains; peak III of strain 15 contained a substantial amount of galactose. Each CPS contained less than 0.2% protein by weight. The significant differences in molecular size and sugar composition among CPSs of these strains of C. neoformans serotype A may partially explain strain differences in virulence and biological properties of the organism.

Ultrastructure of acapsular mutant Cryptococcus neoformans cap 67 and monosaccharide composition of cell extracts.

Acapsular mutant Cryptococcus neoformans cap 67 was grown in Pine's citrate broth medium for 3 days and the cells then transferred to a nitrogen-free medium for 6 days. The cells were subjected to a four stage extraction with buffered Triton-X100, cold dilute alkaline borohydride, hot dilute acetic acid, and a second alkaline extraction. Galactoxylomannan antigens were recovered from the culture supernates of both 3 day-old and 9 day-old yeast cells. The alkaline extracts contained water-soluble galactoxylomannan and a water-insoluble glucan. Dilute acid treatment released a minor amount of carbohydrate from the cells. The second alkaline extraction yielded increased amounts of glucan and galactoxylomannan from the 9 day-old cells. Soluble non-dialyzable cell extracts were antigenically identical in immunodiffusion with the culture supernate antigens. After the extraction sequence, all of the galactose, xylose, and mannose were removed from the cells. The walls retained their shape after extraction but their layers were loosened. Cells resuspended in nitrogen-free medium for six days developed thickened walls with alternating electron-dense and electron-lucent layers. The major constituent of the thickened 9 day-old cell walls was glucose, only 5% glucosamine was detected.

Lipid composition of Cryptococcus neoformans.

The lipid composition of Cryptococcus neoformans grown in Sabouraud's dextrose broth (shake culture) was analysed. The organism contained extremely low amounts of lipid (0.96% dry weight basis) of which 86.1% were nonpolaris lipids, 3.4% phospholipids and the rest were glycolipids and pigments. Alkoxylipids (41%), tryglicerides (18%), diglycerides (7.4%), free fatty acids (5.4%), sterols (4.7%), sterol ester (3.9%) and monoglycerides (2.2%) were found in the nonpolar lipid fraction of C. neoformans. The phospholipid composition (expressed as relative abundance) was: phosphatidylinositol (11.5%), lysophosphatidyl ethanolamine (10.9%), cardiolipin (10.1%), a glycophospholipid (9.5%), lysophosphatidyl choline (4.7%), phosphatidic acid (4.1%), phosphatidyl choline (28.1%), phosphatidyl ethanolamine (14.5%) and an unidentified lipid (6.5%). Phosphatidyl serine, sphingolipids and cerebrosides, generally found in yeast-like fungi, were absent. Probable reasons for the abnormally low lipid content are discussed.

Capsular polysaccharides of Cryptococcus neoformans.

Polysaccharides of Cryptococcus neoformans are considered to have a role in the virulence of this encapsulated fungus. The structure has been determined for the most abundant polysaccharide, a glucuronoxylomannan of varying xylose and ester content. The structural complexity of the capsular material increases from serotype D to A to B to C, but even for the simplest capsule (type D), the immunodeterminants seem to occur only on the side chains. The interactions of some of these groups with antibodies is discussed.

Cryptococcal capsular polysaccharide clearance in nonimmune mice.

Clearance of cryptococcal polysaccharide (CP) from tissues and body fluids of nonimmune mice was studied. Mice were injected intravenously only with one mg of purified CP, and serum, urine and tissues were obtained from each animal at various intervals for a period of 84 days. Tissue extracts, serum and urine were tested for CP content by enzyme-linked immunosorbent assay (ELISA) and latex agglutination. High concentrations of CP were detected by both assays one-half hour after injection in blood (serum), liver, spleen, kidney and lung (extracts). The duration of ELISA detectable CP was longest (70 days) in liver and spleen and shortest (14 days) in lung extract. By 14 days after injection, concentration of CP in the blood fell below that found in the liver and spleen. CP remained detectable (titers 32-64) after all other extracts became negative. These results indicate that CP is stored in tissues (binding mechanism and site unknown), and that the liver and spleen possess greater storage capacity than other tissues. Antibody (IgM) to CP appeared in low titer on the 14th day and thereafter.

Interaction between RNAs from different sources and egg lecithin liposomes.

The interaction of RNAs of molecular weight ranging between 15,000 and 30,000 with egg lecithin large and small unilamellar liposomes has been investigated. Torula and tRNA proved to be incorporated to a similar extent but no linear relationship between molecular weight and percent of incorporation was demonstrated. A relationship between percent of secondary structure of RNA and its ability to be trapped in SUV liposomes is suggested.

Interactions between DNA-binding fluorochromes and mucopolysaccharides in agarose-diffusion test.

Interactions between the two DNA-binding fluorochromes DAPI (4',6-diamidino-2-phenylindole) und Hoechst 33258 (2[2-(4-hydroxy-phenyl)-6-benzimidazolyl]-6-(1-methyl-4-piperazyl)-benzimidazole. 3 HCl) and certain mucopolysaccharides (heparin, sodium dextran sulphate 500, chondroitin sulphate and hyaluronic acid) were demonstrated when applying the agarose diffusion test. The resulting reactions were observed with the naked eye and appeared as precipitation lines. Saponin, a glycoside, was included in these experiments because it led to intensive fluorescent nuclear envelopes when subsequently stained with DAPI. Comparing the two fluorochromes, DAPI achieved stronger reactions with heparin and dextran sulphate 500 whereas the Hoechst fluorochrome led to broader precipitation lines with hyaluronic acid, chondroitin sulphate and saponin. Preliminary experiments also confirmed these interactions at the cellular level.