Cryptococcal glucuronoxylomannan delays translocation of leukocytes across the blood-brain barrier in an animal model of acute bacterial meningitis.

In bacterial meningitis, neurological damage is associated with a high influx of polymorphonuclear leukocytes (PMN) into the brain. Previous data suggest that the capsular component of the fungus C. neoformans, glucuronoxylomannan (GXM), interferes with PMN-migration into the cerebrospinal fluid (CSF). Therefore, a rabbit model of bacterial meningitis was treated intravenously with GXM. This resulted in (1) a reduction of PMN in the CSF at 6 h (P=0.05), (2) reduced peak TNF-alpha concentrations in the CSF, and (3) diminished tissue inflammation and intravascular margination of PMN in GXM-treated animals. Thus, GXM may represent a novel adjuvant anti-inflammatory agent in bacterial meningitis.

Cryptococcus neoformans and its cell wall components induce similar cytokine profiles in human peripheral blood mononuclear cells despite differences in structure.

We studied the cytokine profile of peripheral blood mononuclear cells after stimulation with various cryptococcal strains or its purified cell wall components. After 3 h of stimulation, tumor necrosis factor (TNF) alpha levels were strongly increased, whereas interferon (IFN) gamma and interleukin (IL) 10 levels were increased only slightly, or not at all (respectively). In contrast, after 18 h, TNF-alpha and IFN-gamma levels were (strongly) decreased, whereas the IL-10 levels were increased. The IL-1beta, IL-6 and IL-8 levels were equally high throughout the experiment. In order to establish which of the cryptococcal envelope components contributed most to the observed cytokine profile induced by whole cryptococci, glucuronoxylomannan, galactoxylomannan and mannoproteins were purified and partially characterized biochemically. All cryptococcal components elicited a similar cytokine pattern despite the differences in structure.

Structure of the 13C-enriched O-deacetylated glucuronoxylomannan of Cryptococcus neoformans serotype A determined by NMR spectroscopy.

The complete assignment of 1H and 13C chemical shifts for 99% uniformly 13C-labeled O-deacetylated glucuronoxylomannan (GXM) of Cryptococcus neoformans serotype A isolate 9759-Mu-1 was accomplished by the analysis of HCCH-TOCSY and HCCH-COSY spectra. The sequence of the glycosyl residues was determined by a GHMBC experiment using 20% uniformly 13C-labeled GXM; GXM was prepared by a novel procedure that insured the virtual exclusion of adjacent 13C-labeled carbon atoms. For each residue in the GXM of 9759-Mu-1 we determined its linkage position, its anomeric configuration, and its position in the repeating sequence as follows: [sequence: see text]

Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 10 determined by NMR spectroscopy.

The complete primary structure of the type-specific capsular polysaccharide of Clostridium perfringens Hobbs 10 was determined. The polysaccharide was isolated from C. perfringens Hobbs 10 by cold-water extraction of whole, heavily encapsulated cells. The polysaccharide was purified, by ethanol precipitation, deproteination, selective precipitation with hexadecyltrimethylammonium bromide, ion-exchange chromatography and gel-filtration chromatography. The polysaccharide was comprised of D-glucose, D-galactose, N-acetylgalactosamine, and iduronic acid, in molar ratios of 2:2:1:1. Sequence and linkage assignments of the glycosyl residues were obtained by NMR spectroscopy, specifically by the combination of two-dimensional homonuclear DQF-COSY, TQF-COSY and TOCSY, heteronuclear ¿1H, 13C¿ single-quantum coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC) experiments. The capsular polysaccharide of C. perfringens Hobbs 10 is a polymer composed of a hexasaccharide repeating unit with the following structure: [formula: see text] This structure is novel among bacterial cell-surface polysaccharides, and it is only the second of many serotypically distinct capsular polysaccharides of C. perfringens to be described.

Glucuronoxylomannan of Cryptococcus neoformans serotype B: structural analysis by gas-liquid chromatography-mass spectrometry and 13C-nuclear magnetic resonance spectroscopy.

The major extracellular polysaccharide (glucuronoxylomannan, GXM) from six strains of Cryptococcus neoformans serotype B was characterized by gas-liquid chromatography (g.l.c.), g.l.c.-mass spectrometry (g.l.c.-m.s.), and nuclear magnetic resonance (n.m.r.) spectroscopy. Ultrasonic irradiation (u.i.) was used to reduce the mol.wt. of native GXM from 9.75 x 10(5) to 1.15 x 10(5) without apparent change in its composition (GXM-S). The Xylp:Manp:GlcpA molar ratio of the GXM and GXM-S from the six strains of C. neoformans serotype B is approximately 3.5:3.0:0.6. GXM-S was O-deacetylated (GXM-D) by treatment with NH4OH. The 13C-n.m.r. analysis of GXM-D gave spectra that served as characteristic fingerprints of the structure and also facilitated the assignment of the anomeric carbon resonances to specific structural moieties present in GXM-D. The GXM-D from each serotype B strain was found to be similar by 13C-n.m.r. spectroscopy. The structure contains a linear (1----3)-alpha-D-Manp backbone substituted with 2-O-beta-GlcpA and 2-O-beta-Xylp. beta-Xylp is also O-4 linked to the Manp substituted with GlcpA. In addition, a model for the disposition of the Xylp and GlcpA side chain substituents along the mannopyranan backbone is proposed, based upon results from the combination of g.l.c.-m.s. and 13C-n.m.r. spectroscopy.

Structure of the O-deacetylated glucuronoxylomannan from Cryptococcus neoformans serotype C as determined by 2D 1H NMR spectroscopy.

The primary structure of the O-deacetylated capsular glucuronoxylomannan (GXM) isolated from Cryptococcus neoformans serotype C was investigated by 2D NMR spectroscopy. Assignment of the 1H NMR chemical shifts for the polysaccharide was accomplished from the analysis of DQF-COSY, TOCSY, NOESY and/or ROESY spectra of three isolates (298, 34, and 401). These isolates contain the same polysaccharide glycosyl residues but in different proportions. The serotype C GXM consists of two repeating polysaccharide units that have the following structures: [formula: see text] It is not known if these repeating units comprise a single or two separate polymer chains. The relative amounts of the more highly branched octasaccharide 1 in the isolates studied (i.e., approximately 75% in isolate 34, 50% in isolate 298, and 25% in isolate 401) can be used to explain the serological specificity of these isolates with C. neoformans factor sera, as was previously determined by ELISA in this laboratory. The octasaccharide 1 component is the one previously postulated as the structure of the serotype C GXM although definitive placement of the beta-Xyl-(1-->4) residues had previously not been determined. The heptasaccharide 2 component is uniformly found as the repeating unit in the polysaccharide from serotype B isolates. Additionally, GXM 401 was found to contain a small amount of the hexasaccharide repeating unit usually attributed to serotype A GXM.

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.

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.

Analysis of the common polysaccharide antigens from the cell envelope of Clostridium perfringens type A.

The major, common antigen of Clostridium perfringens type A, isolated and purified independently from three selected strains (Hobbs 5, Hobbs 9, and Hobbs 10), is composed of equimolar amounts of 2-acetamido-2-deoxy-D-mannose (Man-NAc) and 2-acetamido-2-deoxy-D-glucose (GlcNAc). The purified antigen gave a strong immunoprecipitin line by double immunodiffusion in gel. Smith degradation of the major, common antigen caused decomposition of all of the GlcNAc, without concomitant loss in ManNAc, or a perceptible change in serological activity. Therefore, the serological activity of the major, common antigen depended solely on the presence of ManNAc. Data obtained by the 13C-n.m.r.-spectral analysis of the Smith-degradation product revealed that it was a linear-backbone polysaccharide analogous to a Rhodotorula glutinis mannan, but composed of pairs of 2-acetamido-2-deoxymannopyranosyl residues alternately linked beta-(1 leads to 3) and beta-(1 leads to 4). The one-bond, carbon-hydrogen coupling-constant of 162 Hz for both anomeric centers was consistent with the proposed beta-linkages. A similar, 13C-n.m.r.-spectral analysis of the native, common antigen indicated that the GlcNAc residues were randomly connected to three of the four hydroxyl groups not already involved in linking the ManNAc backbone, the 4-hydroxyl group being the exception. A second, serologically inactive, polysaccharide composed of rhamnose, GalNAc, and galactose was identified, but not obtained in homogeneous state. The rhamnosyl residues were probably situated as nonreducing antennae, as they were quantitatively removed by Smith degradation without concomitant decomposition of the polymeric structure of the remaining residues.

Structural variability in the glucuronoxylomannan of Cryptococcus neoformans serotype A isolates determined by 13C NMR spectroscopy.

Cryptococcus neoformans, the etiologic agent of cryptococcal meningoencephalitis, produces glucuronoxylomannan (GXM) as the major capsule component. Purified GXMs obtained from eight serotype A isolates of C. neoformans were treated by ultrasonic irradiation and then O-deacetylated prior to their comprehensive chemical analysis by GLC, GLC-MS, and 13C NMR spectroscopy. The average xylose: mannose: glucuronic acid molar ratio of the eight isolates is 1.96 +/- 0.25: 3.00: 0.58 +/- 0.10. Methylation analyses and 13C NMR spectroscopy show a general structure for GXM that is comprised of a linear (1----3)-alpha-D-mannopyranan substituted with beta-D-GlcpA and with beta-D-Xylp at O-2. Variable quantities of unsubstituted (1----3)-alpha-D-Manp were observed between the eight isolates studied. In several isolates some of the (1----3)-alpha-D-Manp residues are disubstituted with beta-D-GlcpA at O-2 and with beta-D-Xylp at O-4; this type of substitution was not previously thought to occur in serotype A isolates. Heterogeneity, between isolates, in the disposition of the substituents along the mannopyranan backbone was revealed by 13C NMR spectroscopy. The eight isolates, and three isolates previously studied, were each assigned to one of four distinct groups based on the 13C NMR chemical shifts of the anomeric carbons. Six of the eleven isolates gave identical spectra (Group I). The six major anomeric resonances from Group I were assigned to specific glycosidic linkages present in GXM. The remaining five isolates gave more complex spectra that are indicative of additional linkages and comprise the remaining three groups. Three of these five isolates contain substantial amounts of linkages previously thought to be distinctive of serotypes B and C, i.e., Manp residues that are 4-O-glycosylated with beta-D-Xylp. Methylation analyses only predicted an average repeating unit, whereas 13C NMR spectroscopy demonstrated that GXM from each isolate may be categorized into four groups by the occurrence of distinct sequences of carbohydrate residues.

Structural characterization of the galactoxylomannan of Cryptococcus neoformans Cap67.

The galactoxylomannan (GalXM) obtained from the culture supernatant of an acapsular mutant of Cryptococcus neoformans Cap67 was purified by Concanavalin A affinity, ion-exchange, and gel-filtration chromatographies. The structure of GalXM was determined by methylation analysis and by 1D and 2D NMR spectroscopic studies of the intact polysaccharide and of the oligosaccharide fragments generated by Smith degradation and by acetolysis. GalXM is a complex polysaccharide with an alpha-(1-->6) -galactan backbone. The polysaccharide is branched at c-3 of alternate Gal units of the backbone. C-3 is the point of attachment of the oligosaccharide side chains comprised of alpha-D-Man- (1-->3)-alpha-D-Man-(1-->4)- beta-D-Gal-substituted with zero to three terminal beta-Xyl residues as shown in the following structure: [formula: see text].

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).

Polysaccharides from Peptostreptococcus anaerobius and structure of the species-specific antigen.

The cell-envelope antigens of Peptostreptococcus anaerobius were extracted from intact cells by autoclave or alkaline treatment. The purified species-specific antigen (G) was identified among several polysaccharides obtained from the extracts by successive treatments with ribonuclease and pronase followed by ion-exchange and gel-filtration chromatography. G was investigated by 13C- and 31P-n.m.r. spectroscopy, titrimetry, elemental analysis, and gas-liquid chromatography. Oxidation of G with NaIO4 followed by reduction with NaBH4 and mild acid hydrolysis yielded the Smith degradation product of G (GS). Treatment of G and GS with 48% HF gave the respective dephosphorylated products GF and GSF. The structures of GS, GF, and GSF were investigated by 13C-n.m.r. spectroscopy, methylation analysis, and gas-liquid chromatography-mass spectrometry. The principal constituents of G were 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), D-glyceric acid, and phosphate as a diester, in the ratio 2:1:1, and a minor amount of D-glucose (beta-D-Glcp). GS contained D-GlcNAc, D-glyceric acid, glycerol, and phosphate in a 1:1:1:1 ratio. GF and GSF contained D-GlcNAc and D-glyceric acid in the ratios 2:1 and 1:1, respectively. A structure for the principal repeating unit of polymeric G compatible with the analytical data consists of alpha-D-GlcpNAc-(1----3)-alpha-D-GlcpNAc-(1----2)-D-glyceric acid units linked through C-6'-C-6" phosphate diester bridges. This structure is novel for two reasons: (a) unsubstituted glyceric acid residues occur as aglycons in the repeating structure, and (b) phosphate diester bridges link nonanomeric glycose carbons in a non-nucleic acid polymer. The structural role of the minor amount of beta-D-Glcp in G remains unknown.

Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 5 as determined by NMR spectroscopy.

The complete primary structure of the capsular polysaccharide of Clostridium perfringens Hobbs 5, an anaerobic bacterium implicated in food poisoning, was determined. The polysaccharide was isolated from C. perfringens Hobbs 5 cells, after deproteination, by ethanol precipitation and by ion-exchange chromatography. The polysaccharide was comprised of glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, and glucuronic acid, in equimolar ratios. Sequence and linkage assignments of the glycosyl residues were obtained by NMR spectroscopy, specifically by the combination of two-dimensional homonuclear TOCSY and NOESY experiments and heteronuclear (1H, 13C) multiple-quantum coherence (HMQC, HMQC-COSY, HMQC-TOCSY and HMBC) experiments. Thus, the envelope polysaccharide of C. perfringens Hobbs 5 was found to be a polymer composed of a hexasaccharide repeating unit with the following structure: [formula: see text] This structure is novel among bacterial cell-surface polysaccharides, and it is the first of many serotypically distinct capsular polysaccharides of C. perfringens to be described.

Structure of the O-deacetylated glucuronoxylomannan from Cryptococcus neoformans Cap70 as determined by 2D NMR spectroscopy.

Cryptococcus neoformans, an opportunistic pathogen, is the fourth leading cause of death among AIDS patients. The yeast's capsule is a major virulence factor, and serotype is related to the chemical structure of glucuronoxylomannan (GXM), its capsular polysaccharide. The GXM from Cap70, a hypocapsular mutant of serotype D isolate B-3501, was investigated by chemical analysis and 2D NMR spectroscopy. The assignment of 1H and 13C chemical shifts for the O-deacetylated polysaccharide was accomplished from the analysis of DQF-COSY, TOCSY, and gradient-enhanced HSQC spectra. The sequence and linkage positions of glycosyl residues were determined by NOESY and ROESY spectra. Two repeating polysaccharide components were identified as having the following structures in approximately equal proportions: [formula: see text] It is not known if these repeating units comprise a single or two separate polymer chains. Pentasaccharide 2 has been known to be the major GXM polymer of B-3501 and other serotype D isolates. Hexasaccharide 1 is identified for the first time although it has subsequently been identified in other C. neoformans isolates. The presence of 1 in the GXM of Cap70 is consistent with the extra xylose found relative to that in isolate B-3501. The mannose:xylose:glucuronic acid:O-acetyl molar ratio of Cap70 GXM is 3.00:1.73:0.78:1.75, while the same ratio for B-3501 and other serotype D isolates is approximately 3.00:1.00:0.80:1.75. Methylation analysis confirmed that the GXM of Cap70 contains unsubstituted, monosubstituted (2-linked), and disubstituted (2- and 4-linked) mannose in a ratio of 0.87:1.75:0.38. Dot blot immunoassay indicates that Cap70 is a serotype D isolate like its parent strain.

Ventriculoperitoneal shunt malfunction due to migration of the abdominal catheter into the scrotum.

A case is reported in which the peritoneal portion of a ventriculoperitoneal shunt migrated into the scrotum via an indirect inguinal hernia and caused cerebrospinal fluid hydrocele with shunt malfunction.