The capsule of Cryptococcus neoformans

The capsule of Cryptococcus neoformans is its dominant virulence factor and plays a key role in the biology of this fungus. In this essay, we focus on the capsule as a cellular structure and note the limitations inherent in the current methodologies available for its study. Given that no single method can provide the structure of the capsule, our notions of what is the cryptococcal capsule must be arrived at by synthesizing information gathered from very different methodological approaches including microscopy, polysaccharide chemistry and physical chemistry of macromolecules. The emerging picture is one of a carefully regulated dynamic structure that is constantly rearranged as a response to environmental stimulation and cellular replication. In the environment, the capsule protects the fungus against desiccation and phagocytic predators. In animal hosts the capsule functions in both offensive and defensive modes, such that it interferes with immune responses while providing the fungal cell with a defensive shield that is both antiphagocytic and capable of absorbing microbicidal oxidative bursts from phagocytic cells. Finally, we delineate a set of unsolved problems in the cryptococcal capsule field that could provide fertile ground for future investigations

A Glycan FRET Assay for Detection and Characterization of Catalytic Antibodies to the Cryptococcus neoformans Capsule

Classical antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of Cryptococcus neoformans. From this, we designed and synthesized two glycan based Förster Resonance Energy Transfer (FRET) probes, which we used to discover antibodies with innate glycosidase activity and analyse their enzyme kinetics, including mAb 2H1, a polysaccharide lyase, and the most efficient glycosidase to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Further the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labelled by hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing C. neoformans cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages — suggesting the anti-phagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.</p

Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates

<div><p>Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12) cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID) domains (≥100 amino acids) and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases.</p></div

Cellular proteins trapped with Htt polyQ aggregates are disproportionately composed of long intrinsically-disordered (ID) domains.

<p>(A) Comparisons of the percentages of proteins with long ID domains of the 52 yeast proteins in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.t001" target="_blank">Table 1</a> (reproducibly found by TAPI to be tightly associated with Htt-Q103-GFP aggregates) versus 100 randomly-selected yeast proteins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.s002" target="_blank">S1 File</a>). Most of the identified proteins have long ID domains of at least 100 amino acids. (B) Comparisons of the percentages of proteins with long ID domains of the 91 rat proteins in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.t002" target="_blank">Table 2</a> (reproducibly found by TAPI to be tightly associated with GFP-Htt-Q74 aggregates) versus 200 randomly-selected rat proteins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.s003" target="_blank">S2 File</a>). ID domains are defined as regions of 30 or more amino acids with IUPred scores of 0.5 or greater [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.ref047" target="_blank">47</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136362#pone.0136362.ref098" target="_blank">98</a>]. Chi-Square Fisher’s Exact test (Graphpad software) was used to determine significance between TAPI-identified proteins and proteome control sets.</p

Molecular function of proteins associated with Htt-polyQ aggregates as identified by TAPI (N = 91) in <i>Rattus norvegicus</i>.

<p>Molecular function of proteins associated with Htt-polyQ aggregates as identified by TAPI (N = 91) in <i>Rattus norvegicus</i>.</p

Molecular function of proteins associated with Htt-polyQ aggregates as identified by TAPI (N = 52) in <i>Saccharomyces cerevisiae</i>.

<p>Molecular function of proteins associated with Htt-polyQ aggregates as identified by TAPI (N = 52) in <i>Saccharomyces cerevisiae</i>.</p

Htt-polyQ aggregate-associated proteins found in inclusions and aggregates in various neurodegenerative diseases.

<p>Htt-polyQ aggregate-associated proteins found in inclusions and aggregates in various neurodegenerative diseases.</p

The ID domains of Sgt2p and Fus mediate their localization to Htt-polyQ aggregates in yeast cells.

<p>(A, B) Western blots of lysates from yeast strain W303 expressing HttQ25-GFP or HttQ103-GFP in combination with HA-tagged Sgt2p or Sgt2ΔID (A = αGFP; B = αHA). (C, D) Western blots of cells expressing HttQ25-GFP or HttQ103-GFP in combination with FUS or FUSΔID (C = αGFP; D = FUS & α-β actin). Because FUS is quickly degraded in non-denaturing conditions, input controls using urea lysis of cells were included to show initial protein loads. (E, F) Western blots of FUS or FUS(4FL) in HttQ25-GFP-expressing or HttQ103-GFP-expressing cells.</p

Polyglutamine-expanded Huntingtin exon 1 forms aggregates in PC-12 cells that can be isolated by TAPI.

<p>(A) Fluorescence microscopy of PC-12 cells expressing doxycycline inducible transgene GFP-tagged Huntingtin exon 1 (Htt) with normal (Q23) or expanded polygluamine tract (Q74). (B) Western blot of GFP-Htt-Q23 and GFP-Htt-Q74 showing high molecular weight aggregates can be isolated from Htt-Q74 expressing PC-12 cells. Lysate = input; TAPI = purified aggregates.</p