A Glucuronoxylomannan Epitope Exhibits Serotype-Specific Accessibility and Redistributes towards the Capsule Surface during Titanization of the Fungal Pathogen Cryptococcus neoformans

Abstract

Disseminated infections with the fungal species Cryptococcus neoformans or, less frequently, Cryptococcus gattii are an important cause of mortality in immunocompromised individuals. Central to the virulence of both species is an elaborate polysaccharide capsule that consists predominantly of glucuronoxylomannan (GXM). Due to its abundance, GXM is an ideal target for host antibodies, and several monoclonal antibodies (mAbs) have previously been derived using purified GXM or whole capsular preparations as antigens. In addition to their application in the diagnosis of cryptococcosis, anti-GXM mAbs are invaluable tools for studying capsule structure. In this study, we report the production and characterization of a novel anti-GXM mAb, Crp127, that unexpectedly reveals a role for GXM remodeling during the process of fungal titanization. We show that Crp127 recognizes a GXM epitope in an O-acetylation-dependent, but xylosylation-independent, manner. The epitope is differentially expressed by the four main serotypes of Cryptococcus neoformans and C. gattii, is heterogeneously expressed within clonal populations of C. gattii serotype B strains, and is typically confined to the central region of the enlarged capsule. Uniquely, however, this epitope redistributes to the capsular surface in titan cells, a recently characterized morphotype where haploid 5-μm cells convert to highly polyploid cells of >10 μm with distinct but poorly understood capsular characteristics. Titan cells are produced in the host lung and critical for successful infection. Crp127 therefore advances our understanding of cryptococcal morphological change and may hold significant potential as a tool to differentially identify cryptococcal strains and subtypes.

Host environmental conditions induce small fungal cell size and alter population heterogeneity in Cryptococcus neoformans

“…The C. neoformans cell wall is covered by a protective capsule, which not only impairs phagocytosis by macrophages, but also mask PAMPs from being engaged by host PRRs (53). Moreover, H99 titan cells have been reported to have altered capsule compared to yeast (5, 54). We have previously demonstrated that Crp127 is an anti-capsule monoclonal IgM (Immunoglobulin M) antibody associated with O-mannosylation, to capsular polysaccharide and that its localization is dynamic throughout titan development (54).…”

“…Moreover, H99 titan cells have been reported to have altered capsule compared to yeast (5, 54). We have previously demonstrated that Crp127 is an anti-capsule monoclonal IgM (Immunoglobulin M) antibody associated with O-mannosylation, to capsular polysaccharide and that its localization is dynamic throughout titan development (54). We therefore assessed the capsule of Zc isolates after titan induction.…”

“…We therefore assessed the capsule of Zc isolates after titan induction. Induced cells were stained with both India ink to reveal overall capsule size and Crp127 IgM antibody to reveal localization of the Crp127 epitope (Figure 6A,B) (54). All the isolates expressed robust capsule and were reactive to Crp127 IgM, consistent with our previous reports (Figure 6A).…”

“…While the fungal cell wall PAMPs β-glucan and mannan have been demonstrated to trigger pro-inflammatory immune response via C-type lectin receptors, the C. neoformans capsule masks these PAMPs from the host, and the major ligand for C. neoformans recognition remains unclear (65). The dynamic nature of the titan cell wall, particularly chitin, and capsule relative to yeast suggest a complex strategy for host evasion (12, 66). We further demonstrate here that the penetrance of titanization within a population can impact the strength of the host response: purified H99 titans are potent TNF inducers, while mixed cultures are less stimulatory.…”

Steryl Glycosides in Fungal Pathogenesis: An Understudied Immunomodulatory Adjuvant

“…Titan cell formation and the role of SGs found in extracellular vesicles (EVs) are important features that can be tied into how the fungal cells respond and trigger protection in the host. Titanization is the enlargement of the surrounding capsule of C. neoformans when grown in minimal media or the hostile host environment [90][91][92]. Since SGs are found in the plasma membrane, the multi-fold enlargement of the capsule may hinder or ablate the physical properties SGs convey to the capsule, but this is purely speculation, since this has not been investigated as well and is not currently under investigation by our group.…”