O-Acetylation of Cryptococcal Capsular Glucuronoxylomannan Is Essential for Interference with Neutrophil Migration

Abstract

The capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans has been shown to interfere with neutrophil migration. Although several receptors have been implied to mediate this process, the structural perspectives are unknown. Here, we assess the contribution of 6-O-acetylation and xylose substitution of the (1→3)-α-d-mannan backbone of GXM, the variable structural features of GXM, to the interference with neutrophil migration. We compare chemically deacetylated GXM and acetyl- or xylose-deficient GXM from genetically modified strains with wild-type GXM in their ability to inhibit the different phases of neutrophil migration. Additionally, we verify the effects of de-O-acetylation on neutrophil migration in vivo. De-O-acetylation caused a dramatic reduction of the inhibitory capacity of GXM in the in vitro assays for neutrophil chemokinesis, rolling on E-selectin and firm adhesion to endothelium. Genetic removal of xylose only marginally reduced the ability of GXM to reduce firm adhesion. In vivo, chemical deacetylation of GXM significantly reduced its ability to interfere with neutrophil recruitment in a model of myocardial ischemia (65% reduction vs a nonsignificant reduction in tissue myeloperoxidase, respectively). Our findings indicate that 6-O-acetylated mannose of GXM is a crucial motive for the inhibition of neutrophil recruitment.

Cryptococcus gattiiIsolates from the British Columbia Cryptococcosis Outbreak Induce Less Protective Inflammation in a Murine Model of Infection thanCryptococcus neoformans

“…Neutrophil infiltration during pulmonary infection. Neutrophils are important mediators of the protective immune response against C. neoformans (18,35,46), and as described above, our flow cytometry analysis revealed differences in neutrophil accumulation for the C. gattii and C. neoformans infections. To investigate this phenomenon in more detail, we measured neutrophil infiltration in the lungs of infected C57BL/6 mice by cytological and enzymatic methods.…”

“…Conversely, the lack of protective inflammation in C. gattii-infected mice may be due to a smaller capsule size in vivo. The capsule also may play an important role in interfering with neutrophil migration because, for example, 6-O-acetyl groups on the major component of the capsule, glucuronoxylmannan (GXM), are responsible for the inhibition of neutrophil migration in vitro (18,20). Furthermore, free GXM has been shown to promote L-selectin shedding from neutrophils, and this may interfere with migration (17).…”

Extracellular Vesicles from Cryptococcus neoformans Modulate Macrophage Functions

“…neoformans produces several exocellular antigens with apparently conflicting functions with regard to pathogenesis. For instance, GXM, the major cryptococcal virulence factor, protects the fungus against phagocytosis and inhibits leukocyte migration (17,21,22,34). However, this polysaccharide, in its capsular form, is also an efficient inducer of the activation of the alternative pathway of the complement system, which results in the deposition of opsonins in the capsule and increased phagocytosis (33,35).…”

The Physical Properties of the Capsular Polysaccharides from Cryptococcus neoformans Suggest Features for Capsule Construction

“…Although the mechanisms responsible for these effects are not well understood, GXM can interact with the cellular receptor CD14, and Toll-like receptors 2 and 4 (13). Binding of Toll-like receptor 4 appears critical for inhibiting neutrophil migration and the up-regulation of Fas ligand on macrophages (14,15). GXM has attracted interest as a vaccine component and a target for antibody-based therapy since antibody to GXM is protective in animal models.…”

“…The GXM of individual strains of C. neoformans differ in their ratios of the repeating units, and recent oligosaccharide compositional analysis suggests the occurrence of non-uniform side group addition and variability in co-polymerization (21). 5 Alterations in GXM composition have been experimentally correlated with changes in C. neoformans virulence, inhibition of neutrophil migration, and antibody recognition (15,(23)(24)(25)(26)(27).…”