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

Sheng, Shuqun; Cherniak, Robert

doi:10.1016/s0008-6215(97)00084-0

Cited by 44 publications

(32 citation statements)

References 31 publications

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“…By comparison to the assignments made in this previous study, we were able to assign each crosspeak detected in the H99 spectra to the one glucuronic acid, two xylose, and three mannose residues that form the predominant repeating unit of the C. neoformans serotype A GXM (as shown in Fig. 1A) (30).…”

Section: Vol 6 2007 Parallel ␤-Helix Proteins In C Neoformans 633mentioning

confidence: 81%

“…Purified exopolysaccharide was obtained by a protocol adapted from previously described methods (11,30). C. neoformans strains were grown in 100 ml of liquid YNB medium for 5 days.…”

Section: Methodsmentioning

confidence: 99%

“…For serotype A, the GXM polymer is composed primarily of a repeating mannose triplet (␣-1,3 linked) with glucuronic acid 132 linked to the first mannose of the triplet and xylose 132 linked to the second and third mannoses (Fig. 1A) (30).…”

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confidence: 99%

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Parallel β-Helix Proteins Required for Accurate Capsule Polysaccharide Synthesis and Virulence in the Yeast Cryptococcus neoformans

Liu¹,

Chow²,

Madhani³

2007

Eukaryot Cell

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The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an ␣-1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel ␤-helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry-colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional nuclear magnetic resonance spectroscopy of purified GXM from the mutants indicated that both the wild-type GXM structure and novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wild-type capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. PBX1 and PBX2 knockout mutants showed severely attenuated virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered.

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Section: Vol 6 2007 Parallel ␤-Helix Proteins In C Neoformans 633mentioning

confidence: 81%

“…Purified exopolysaccharide was obtained by a protocol adapted from previously described methods (11,30). C. neoformans strains were grown in 100 ml of liquid YNB medium for 5 days.…”

Section: Methodsmentioning

confidence: 99%

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Parallel β-Helix Proteins Required for Accurate Capsule Polysaccharide Synthesis and Virulence in the Yeast Cryptococcus neoformans

Liu¹,

Chow²,

Madhani³

2007

Eukaryot Cell

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“…The smallest repeating moieties (Fig. 6) were determined by the assessment of connectivity between carbohydrate residues, using ( 1 H, 13 C) HMBC spectra and the chemical shifts in mutant strains compared to that of the parental strain as well as to those of previous work (1,27,37,41). The structure of the GXM isolated from the parental strain was identical to that of the chemotype Chem1, which is dominant in serotype D isolates (12, 43) (moiety 1 in Fig.…”

Section: Moyrand Et Al Eukaryot Cellmentioning

confidence: 89%

Cas3p Belongs to a Seven-Member Family of Capsule Structure Designer Proteins

et al. 2004

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The polysaccharide capsule is the main virulence factor of the basidiomycetous yeast Cryptococcus neoformans. Four genes (CAP10, CAP59, CAP60, and CAP64) essential for capsule formation have been previously identified, although their roles in the biosynthetic pathway remain unclear. A genetic and bioinformatics approach allowed the identification of six CAP64-homologous genes, named CAS3, CAS31, CAS32, CAS33, CAS34, and CAS35, in the C. neoformans genome. This gene family is apparently specific in a subclass of the basidiomycete fungi. Single as well as double deletions of these genes in all possible combinations demonstrated that none of the CAP64-homologous genes were essential for capsule formation, although the cas35⌬ strains displayed a hypocapsular phenotype. The chemical structure of the glucuronomannan (GXM) produced by the CAS family deletants revealed that these genes determined the position and the linkage of the xylose and/or O-acetyl residues on the mannose backbone. Hence, these genes are all involved in assembly of the GXM structure in C. neoformans.

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“…The cultures were autoclaved and centrifuged at 18,000 ϫ g for 1 h at RT, and the supernatant fraction was filtered through a 0.22-m-pore-size mem-brane filter and used for GXM purification. Purification, based on precipitation of the negatively charged GXM with the cationic detergent cetyltrimethylammonium bromide (CTAB), was performed as described previously (22,44).…”

Section: Methodsmentioning

confidence: 99%

Pbx Proteins in Cryptococcus neoformans Cell Wall Remodeling and Capsule Assembly

et al. 2014

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The cryptococcal capsule is a critical virulence factor of an important pathogen, but little is known about how it is associated with the cell or released into the environment. Two mutants lacking PBX1 and PBX2 were found to shed reduced amounts of the capsule polysaccharide glucuronoxylomannan (GXM). Nuclear magnetic resonance, composition, and physical analyses showed that the shed material was of normal mass but was slightly enriched in xylose. In contrast to previous reports, this material contained no glucose. Notably, the capsule fibers of pbx⌬ mutant cells grown under capsule-inducing conditions were present at a lower than usual density and were loosely attached to the cell wall. Mutant cell walls were also defective, as indicated by phenotypes including abnormal cell morphology, reduced mating filamentation, and altered cell integrity. All observed phenotypes were shared between the two mutants and exacerbated in a double mutant. Consistent with a role in surface glycan synthesis, the Pbx proteins localized to detergent-resistant membrane domains. These results, together with the sequence motifs in the Pbx proteins, suggest that Pbx1 and Pbx2 are redundant proteins that act in remodeling the cell wall to maintain normal cell morphology and precursor availability for other glycan synthetic processes. Their absence results in aberrant cell wall growth and metabolic imbalance, which together impact cell wall and capsule synthesis, cell morphology, and capsule association. The surface changes also lead to increased engulfment by host phagocytes, consistent with the lack of virulence of pbx mutants in animal models.

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

Cited by 44 publications

References 31 publications

Parallel β-Helix Proteins Required for Accurate Capsule Polysaccharide Synthesis and Virulence in the Yeast Cryptococcus neoformans

Parallel β-Helix Proteins Required for Accurate Capsule Polysaccharide Synthesis and Virulence in the Yeast Cryptococcus neoformans

Cas3p Belongs to a Seven-Member Family of Capsule Structure Designer Proteins

Pbx Proteins in Cryptococcus neoformans Cell Wall Remodeling and Capsule Assembly

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