scholarly journals Binding of the Wheat Germ Lectin to Cryptococcus neoformans Suggests an Association of Chitinlike Structures with Yeast Budding and Capsular Glucuronoxylomannan

2007 ◽  
Vol 7 (4) ◽  
pp. 602-609 ◽  
Author(s):  
Marcio L. Rodrigues ◽  
Mauricio Alvarez ◽  
Fernanda L. Fonseca ◽  
Arturo Casadevall
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Wheat Germ ◽  
Capsular Polysaccharide ◽  
Complex Structure ◽  
Anion Exchange Chromatography ◽  
Sialic Acids ◽  
Exchange Chromatography ◽  
Wheat Germ Lectin ◽  
Competition Assays

ABSTRACT The capsule of Cryptococcus neoformans is a complex structure whose assembly requires intermolecular interactions to connect its components into an organized structure. In this study, we demonstrated that the wheat germ agglutinin (WGA), which binds to sialic acids and β-1,4-N-acetylglucosamine (GlcNAc) oligomers, can also bind to cryptococcal capsular structures. Confocal microscopy demonstrated that these structures form round or hooklike projections linking the capsule to the cell wall, as well as capsule-associated structures during yeast budding. Chemical analysis of capsular extracts by gas chromatography coupled to mass spectrometry and high-pH anion-exchange chromatography suggested that the molecules recognized by WGA were firmly associated with the cell wall. Enzymatic treatment, competition assays, and staining with chemically modified WGA revealed that GlcNAc oligomers, but not sialic acids, were the molecules recognized by the lectin. Accordingly, treatment of C. neoformans cells with chitinase released glucuronoxylomannan (GXM) from the cell surface and reduced the capsule size. Chitinase-treated acapsular cells bound soluble GXM in a modified pattern. These results indicate an association of chitin-derived structures with GXM and budding in C. neoformans, which may represent a new mechanism by which the capsular polysaccharide interacts with the cell wall and is rearranged during replication.

scholarly journals Capsular Polysaccharide Production in Enterococcus faecalis and Contribution of CpsF to Capsule Serospecificity

2009 ◽  
Vol 191 (20) ◽  
pp. 6203-6210 ◽  
Author(s):  
Lance R. Thurlow ◽  
Vinai Chittezham Thomas ◽  
Lynn E. Hancock
Keyword(s):  
Enterococcus Faecalis ◽  
Capsular Polysaccharide ◽  
Bacterial Species ◽  
Amperometric Detection ◽  
Genetic System ◽  
Lipoteichoic Acid ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Detection Analysis ◽  
Capsule Production

ABSTRACT Many bacterial species produce capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system. The gram-positive pathogen Enterococcus faecalis was previously reported to produce one of four capsule serotypes (A, B, C, or D). Previous studies describing the four capsule serotypes of E. faecalis were based on immunodetection methods; however, the underlying genetics of capsule production did not fully support these findings. Previously, it was shown that capsule production for serotype C (Maekawa type 2) was dependent on the presence of nine open reading frames (cpsC to cpsK). Using a novel genetic system, we demonstrated that seven of the nine genes in the cps operon are essential for capsule production, indicating that serotypes A and B do not make a capsular polysaccharide. In support of this observation, we showed that serotype C and D capsule polysaccharides mask lipoteichoic acid from detection by agglutinating antibodies. Furthermore, we determined that the genetic basis for the difference in antigenicity between serotypes C and D is the presence of cpsF in serotype C strains. High-pH anion-exchange chromatography with pulsed amperometric detection analysis of serotype C and D capsules indicated that cpsF is responsible for glucosylation of serotype C capsular polysaccharide in E. faecalis.

scholarly journals Cell-wall polysaccharides and glycoproteins of parenchymatous tissues of runner bean (Phaseolus coccineus)

1990 ◽  
Vol 269 (2) ◽  
pp. 393-402 ◽  
Author(s):  
P Ryden ◽  
R R Selvendran
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Anion Exchange Chromatography ◽  
Structural Features ◽  
Good Evidence ◽  
Exchange Chromatography ◽  
Cell Wall Polysaccharides ◽  
Pectic Polysaccharides ◽  
Runner Bean ◽  
Cellulose Residue

1. Polymers were solubilized from the cell walls of parenchyma from mature runner-bean pods with minimum degradation by successive extractions with cyclohexane-trans-1,2-diamine-NNN′N′-tetra-acetate (CDTA), Na2CO3 and KOH to leave the alpha-cellulose residue, which contained cross-linked pectic polysaccharides and Hyp-rich glycoproteins. These were solubilized with chlorite/acetic acid and cellulase. The polymers were fractionated by anion-exchange chromatography, and fractions were subjected to methylation analysis. 2. The pectic polysaccharides differed in their ease of extraction, and a small proportion were highly cross-linked. The bulk of the pectic polysaccharides solubilized by CDTA and Na2CO3 were less branched than those solubilized by KOH. There was good evidence that most of the pectic polysaccharides were not degraded during extraction. 3. The protein-containing fractions included Hyp-rich and Hyp-poor glycoproteins associated with easily extractable pectic polysaccharides, Hyp-rich glycoproteins solubilized with 4M-KOH+borate, the bulk of which were not associated with pectic polysaccharides, and highly cross-linked Hyp-rich glycoproteins. 4. Isodityrosine was not detected, suggesting that it does not have a (major) cross-linking role in these walls. Instead, it is suggested that phenolics, presumably linked to C-5 of 3,5-linked Araf residues of Hyp-rich glycoproteins, serve to cross-link some of the polymers. 5. There were two main types of xyloglucan, with different degrees of branching. The bulk of the less branched xyloglucans were solubilized by more-concentrated alkali. The anomeric configurations of the sugars in one of the highly branched xyloglucans were determined by 13C-n.m.r. spectroscopy. 6. The structural features of the cell-wall polymers and complexes are discussed in relation to the structure of the cell walls of parenchyma tissues.

scholarly journals Lipophilic Dye Staining of Cryptococcus neoformans Extracellular Vesicles and Capsule

2009 ◽  
Vol 8 (9) ◽  
pp. 1373-1380 ◽  
Author(s):  
André Moraes Nicola ◽  
Susana Frases ◽  
Arturo Casadevall
Keyword(s):  
Cryptococcus Neoformans ◽  
Extracellular Vesicles ◽  
Capsular Polysaccharide ◽  
Complex Structure ◽  
Spectral Characteristics ◽  
Acid Dyes ◽  
Whole Cells ◽  
Direct Binding

ABSTRACT Cryptococcus neoformans is an encapsulated yeast that causes systemic mycosis in immunosuppressed individuals. Recent studies have determined that this fungus produces vesicles that are released to the extracellular environment both in vivo and in vitro. These vesicles contain assorted cargo that includes several molecules associated with virulence and implicated in host-pathogen interactions, such as capsular polysaccharides, laccase, urease, and other proteins. To date, visualization of extracellular vesicles has relied on transmission electron microscopy, a time-consuming technique. In this work we report the use of fluorescent membrane tracers to stain lipophilic structures in cryptococcal culture supernatants and capsules. Two dialkylcarbocyanine probes with different spectral characteristics were used to visualize purified vesicles by fluorescence microscopy and flow cytometry. Dual staining of vesicles with dialkylcarbocyanine and RNA-selective nucleic acid dyes suggested that a fraction of the vesicle population carried RNA. Use of these dyes to stain whole cells, however, was hampered by their possible direct binding to capsular polysaccharide. A fluorescent phospholipid was used as additional membrane tracer to stain whole cells, revealing punctate structures on the edge of the capsule which are consistent with vesicular trafficking. Lipophilic dyes provide new tools for the study of fungal extracellular vesicles and their content. The finding of hydrophobic regions in the capsule of C. neoformans adds to the growing evidence for a structurally complex structure composed of polysaccharide and nonpolysaccharide components.

scholarly journals Chitinases Are Essential for Sexual Development but Not Vegetative Growth in Cryptococcus neoformans

2009 ◽  
Vol 8 (11) ◽  
pp. 1692-1705 ◽  
Author(s):  
Lorina G. Baker ◽  
Charles A. Specht ◽  
Jennifer K. Lodge
Keyword(s):  
Cell Wall ◽  
Sexual Reproduction ◽  
Cryptococcus Neoformans ◽  
Vegetative Growth ◽  
Cell Walls ◽  
Cell Structure ◽  
Complex Structure ◽  
Hydrolytic Enzymes ◽  
Fungal Cell ◽  
Fungal Cell Walls

ABSTRACT Cryptococcus neoformans is an opportunistic pathogen that mainly infects immunocompromised individuals. The fungal cell wall of C. neoformans is an excellent target for antifungal therapies since it is an essential organelle that provides cell structure and integrity. Importantly, it is needed for localization or attachment of known virulence factors, including melanin, phospholipase, and the polysaccharide capsule. The polysaccharide fraction of the cryptococcal cell wall is a complex structure composed of chitin, chitosan, and glucans. Chitin is an indispensable component of many fungal cell walls that contributes significantly to cell wall strength and integrity. Fungal cell walls are very dynamic, constantly changing during cell division and morphogenesis. Hydrolytic enzymes, such as chitinases, have been implicated in the maintenance of cell wall plasticity and separation of the mother and daughter cells at the bud neck during vegetative growth in yeast. In C. neoformans we identified four predicted endochitinases, CHI2, CHI21, CHI22, and CHI4, and a predicted exochitinase, hexosaminidase, HEX1. Enzymatic analysis indicated that Chi2, Chi22, and Hex1 actively degraded chitinoligomeric substrates. Chi2 and Hex1 activity was associated mostly with the cellular fraction, and Chi22 activity was more prominent in the supernatant. The enzymatic activity of Hex1 increased when grown in media containing only N-acetylglucosamine as a carbon source, suggesting that its activity may be inducible by chitin degradation products. Using a quadruple endochitinase deletion strain, we determined that the endochitinases do not affect the growth or morphology of C. neoformans during asexual reproduction. However, mating assays indicated that Chi2, Chi21, and Chi4 are each involved in sexual reproduction. In summary, the endochitinases were found to be dispensable for routine vegetative growth but not sexual reproduction.

scholarly journals Cell Wall Calcium and Hemicellulose Have a Role in the Fruit Firmness during Storage of Blueberry (Vaccinium spp.)

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 553
Author(s):  
Patricio Olmedo ◽  
Baltasar Zepeda ◽  
Bárbara Rojas ◽  
Christian Silva-Sanzana ◽  
Joaquín Delgado-Rioseco ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cold Storage ◽  
Calcium Binding ◽  
High Performance ◽  
Calcium Concentration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fruit Firmness ◽  
Cell Wall Components ◽  
Wall Components

The firmness of blueberry is one of its most significant quality attributes. Modifications in the composition of the cell wall have been associated with changes in the fruit firmness. In this work, cell wall components and calcium concentration in two blueberry cultivars with contrasting firmness phenotypes were evaluated at harvest and 30 days cold storage (0 °C). High performance anion-exchange chromatography with pulse amperometric detector (HPAEC-PAD) analysis was performed using the “Emerald” (firmer) and “Jewel” (softer) blueberry cultivars, showing increased glucose in the firmer cultivar after cold storage. Moreover, the LM15 antibody, which recognizes xyloglucan domains, displayed an increased signal in the Emerald cultivar after 30 d cold storage. Additionally, the antibody 2F4, recognizing a homogalacturonan calcium-binding domain, showed a greater signal in the firmer Emerald blueberries, which correlates with a higher calcium concentration in the cell wall. These findings suggest that xyloglucan metabolism and a higher concentration of cell wall calcium influenced the firmness of the blueberry fruit. These results open new perspectives regarding the role of cell wall components as xyloglucans and calcium in blueberry firmness.

scholarly journals Cryptococcus neoformans capsule regrowth experiments reveal dynamics of enlargement and architecture

2021 ◽  
Author(s):  
Maggie P. Wear ◽  
Ella Jacobs ◽  
Siqing Wang ◽  
Scott McConnell ◽  
Anthony Bowen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Virulence Factor ◽  
Fungal Pathogen ◽  
Capsular Polysaccharide ◽  
Wall Surface ◽  
Polysaccharide Capsule ◽  
Capsular Material ◽  
Enzymatic Methods

The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded characterization. Polysaccharides remain attached to the cell as capsular polysaccharide (CPS) or are shed into the surroundings in the form of exopolysaccharide (EPS). While a great deal of study has been done examining the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and removed capsular material. Sonication and glucanex digestion yield soluble CPS preparations, while French Press and modified glucanex digestion plus vortexing remove the capsule and cell wall producing polysaccharide aggregates that we call capsule ghosts. The existence of capsule ghosts implies an inherent organization that allows it to exist independent of the cell wall surface. As sonication and glucanex digestion were noncytotoxic, it was possible to observe the cryptococcal cells rebuilding their capsule, revealing new insights into capsule architecture and synthesis consistent with a model in which the capsule is assembled from smaller polymers, which are then assemble into larger ones.

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