Methods of Cryptococcal Polysaccharide Analysis Using ELISA.

One of the standard assays for the fungal pathogen Cryptococcus neoformans is the glucuronoxylomannan (GXM) ELISA. This assay utilizes monoclonal antibodies targeted against the critical virulence factor, the polysaccharide (PS) capsule. GXM ELISA is one of the most used assays in the field used for diagnosis of cryptococcal infection, quantification of PS content, and determination of binding specificity for antibodies. Here we present three variations of the GXM ELISA used by our group-indirect, capture, and competition ELISAs. We have also provided some history, perspective, and notes on these methods, which we hope will help the reader choose, and implement, the best assay for their research.While it has long been referred to as the GXM ELISA, we also suggest a name update to better reflect our updated understanding of the polysaccharide antigens targeted by this assay. The Cryptococcal PS ELISA is a more accurate description of this set of methodologies and the antigens they measure. Finally, we discuss the limitations of this assay and put forth future plans for expanding the antigens assayed by ELISA.

Semisynthetic Glycoconjugate Vaccine Candidates against Cryptococcus neoformans.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, which poses a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semisynthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semisynthetic glycoconjugate vaccines contain an identical synthetic decasaccharide (M2 motif) antigen. This antigen is present in serotype A strains, which constitute 95% of the clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity toward M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced weakly opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). These findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. This antigen could serve as a component in a multivalent GXM motif vaccine.

Semi-synthetic glycoconjugate vaccine candidate against Cryptococcus neoformans.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, posing a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semi-synthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semi-synthetic glycoconjugate vaccines contain the identical synthetic decasaccharide (M2 motif) antigen. This motif is present in serotype A strains, which constitute 95% of clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity towards M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). While these findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. It could serve as a component in a multi-valent GXM motif vaccine, enhancing both strength and breadth of immune responses.

The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: A combined NMR and MD study.

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans' capsular polysaccharide and its shed exopolysaccharide function both as key virulence factors and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this study, NOE and J-coupling values from NMR experiments were consistent with a converged structure of the synthetic decasaccharide, GXM10-Ac3, calculated from MD simulations. GXM10-Ac3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is a hexasaccharide with a three-residue α-mannan backbone, modified by ß-(1→2)-xyloses (Xyl) on the first two mannoses (Man) and a ß-(1→2)-glucuronic acid (GlcA) on the third Man. Combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with Xyl/GlcA branches alternating sides along the α-mannan backbone. O-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac3 structure is uniformly represented throughout the polysaccharide. This derived GXM model displays high flexibility while maintaining a structural identity, yielding insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.

Synthetic Glycans Reveal Determinants of Antibody Functional Efficacy against a Fungal Pathogen.

Antibodies play a vital role in the immune response to infectious diseases and can be administered passively to protect patients. In the case of Cryptococcus neoformans, a WHO critical priority fungal pathogen, infection results in antibodies targeting capsular glucuronoxylomannan (GXM). These antibodies yield protective, non-protective, and disease-enhancing outcomes when administered passively. However, it was unknown how these distinct antibodies recognized their antigens at the molecular level, leading to the hypothesis that they may target different GXM epitopes. To test this hypothesis, we constructed a microarray containing 26 glycans representative of those found in highly virulent cryptococcal strains and utilized it to study 16 well-characterized monoclonal antibodies. Notably, we found that protective and non-protective antibodies shared conserved reactivity to the M2 motif of GXM, irrespective of the strain used in infection or GXM-isolated to produce a conjugate vaccine. Here, only two antibodies, 12A1 and 18B7, exhibited diverse trivalent GXM motif reactivity. IgG antibodies associated with protective responses showed cross-reactivity to at least two GXM motifs. This molecular understanding of antibody binding epitopes was used to map the antigenic diversity of two Cryptococcus neoformans strains, which revealed the exceptional complexity of fungal capsular polysaccharides. A multi-GXM motif vaccine holds the potential to effectively address this antigenic diversity. Collectively, these findings underscore the context-dependent nature of antibody function and challenge the classification of anti-GXM epitopes as either "protective" or "non-protective".

The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: A combined NMR and MD study.

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans capsular polysaccharide and shed exopolysaccharide functions both as a key virulence factor and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this combined NMR and MD study, experimentally determined NOEs and J-couplings support a structure of the synthetic decasaccharide, GXM10-Ac3, obtained by MD. GXM10-Ac3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is characterized by a 6-residue α-mannan backbone repeating unit, consisting of a triad of α-(1→3)-mannoses, modified by ß-(1→2)-xyloses on the first two mannoses and a ß-(1→2)-glucuronic acid on the third mannose. The combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with xylose/glucuronic acid branches alternating sides along the α-mannan backbone. O-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac3 structure is uniformly represented throughout the polysaccharide. This experimentally consistent GXM model displays high flexibility while maintaining a structural identity, yielding new insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.

Spike-protein proteolytic antibodies in COVID-19 convalescent plasma contribute to SARS-CoV-2 neutralization.

Understanding the mechanisms of antibody-mediated neutralization of SARS-CoV-2 is critical in combating the COVID-19 pandemic. Based on previous reports of antibody catalysis, we investigated the proteolysis of spike (S) by antibodies in COVID-19 convalescent plasma (CCP) and its contribution to viral neutralization. Quenched fluorescent peptides were designed based on S epitopes to sensitively detect antibody-mediated proteolysis. We observed epitope cleavage by CCP from different donors which persisted when plasma was heat-treated or when IgG was isolated from plasma. Further, purified CCP antibodies proteolyzed recombinant S domains, as well as authentic viral S. Cleavage of S variants suggests CCP antibody-mediated proteolysis is a durable phenomenon despite antigenic drift. We differentiated viral neutralization occurring via direct interference with receptor binding from that occurring by antibody-mediated proteolysis, demonstrating that antibody catalysis enhanced neutralization. These results suggest that antibody-catalyzed damage of S is an immunologically relevant function of neutralizing antibodies against SARS-CoV-2.

Cuticular profiling of insecticide resistant Aedes aegypti.

Insecticides have made great strides in reducing the global burden of vector-borne disease. Nonetheless, serious public health concerns remain because insecticide-resistant vector populations continue to spread globally. To circumvent insecticide resistance, it is essential to understand all contributing mechanisms. Contact-based insecticides are absorbed through the insect cuticle, which is comprised mainly of chitin polysaccharides, cuticular proteins, hydrocarbons, and phenolic biopolymers sclerotin and melanin. Cuticle interface alterations can slow or prevent insecticide penetration in a phenomenon referred to as cuticular resistance. Cuticular resistance characterization of the yellow fever mosquito, Aedes aegypti, is lacking. In the current study, we utilized solid-state nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry, and transmission electron microscopy to gain insights into the cuticle composition of congenic cytochrome P450 monooxygenase insecticide resistant and susceptible Ae. aegypti. No differences in cuticular hydrocarbon content or phenolic biopolymer deposition were found. In contrast, we observed cuticle thickness of insecticide resistant Ae. aegypti increased over time and exhibited higher polysaccharide abundance. Moreover, we found these local cuticular changes correlated with global metabolic differences in the whole mosquito, suggesting the existence of novel cuticular resistance mechanisms in this major disease vector.

Cuticular profiling of insecticide resistant Aedes aegypti.

Insecticides have made great strides in reducing the global burden of vector-borne disease. Nonetheless, serious public health concerns remain because insecticide-resistant vector populations continue to spread globally. To circumvent insecticide resistance, it is essential to understand all contributing mechanisms. Contact-based insecticides are absorbed through the insect cuticle, which is comprised mainly of chitin polysaccharides, cuticular proteins, hydrocarbons, and phenolic biopolymers sclerotin and melanin. Cuticle interface alterations can slow or prevent insecticide penetration in a phenomenon referred to as cuticular resistance. Cuticular resistance characterization of the yellow fever mosquito, Aedes aegypti , is lacking. In the current study, we utilized solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy, gas chromatography/mass spectrometry (GC-MS), and transmission electron microscopy (TEM) to gain insights into the cuticle composition of congenic cytochrome P450 monooxygenase insecticide resistant and susceptible Ae. aegypti . No differences in cuticular hydrocarbon content or phenolic biopolymer deposition were found. In contrast, we observed cuticle thickness of insecticide resistant Ae. aegypti increased over time and exhibited higher polysaccharide abundance. Moreover, we found these local cuticular changes correlated with global metabolic differences in the whole mosquito, suggesting the existence of novel cuticular resistance mechanisms in this major disease vector.

Similar evolutionary trajectories in an environmental Cryptococcus neoformans isolate after human and murine infection.

A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from an immunocompromised patient with cryptococcosis based on molecular analyses available in 2000. Here, we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MATα strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including eight nonsynonymous changes involving seven genes. To ascertain whether changes in these genes are selected for during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frameshift mutation in one of the seven genes altered in the human sample (LQVO5_000317), a gene predicted to encode an SWI-SNF chromatin-remodeling complex protein. In addition, both cockatoo and patient strains as well as mouse-passaged isolates obtained from brain tissue had a premature stop codon in a homologue of ZFC3 (LQVO5_004463), a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and reverted to a full-length protein in the mouse-passaged isolates obtained from lung tissue. The patient strain and mouse-passaged isolates show variability in virulence factors, with differences in capsule size, melanization, rates of nonlytic expulsion from macrophages, and amoeba predation resistance. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

Lyophilization induces physicochemical alterations in cryptococcal exopolysaccharide.

Microbial polysaccharide characterization requires purification that often involves detergent precipitation and lyophilization. Here we examined physicochemical changes following lyophilization of Cryptococcus neoformans exopolysaccharide (EPS). Solution 1H Nuclear Magnetic Resonance (NMR) reveals significant anomeric signal attenuation following lyophilization of native EPS while 1H solid-state Nuclear Magnetic Resonance (ssNMR) shows few changes, suggesting diminished molecular motion and consequent broadening of 1H NMR polysaccharide resonances. 13C ssNMR, dynamic light scattering, and transmission electron microscopy show that, while native EPS has rigid molecular characteristics and contains small, loosely packed polysaccharide assemblies, lyophilized and resuspended EPS is disordered and contains larger dense aggregates, suggesting that structural water molecules in the interior of the polysaccharide assemblies are removed during extensive lyophilization. Importantly, mAbs to C. neoformans polysaccharide bind native EPS more strongly than lyophilized EPS. Together, these observations argue for caution when interpreting the biological and immunological attributes of polysaccharides that have been lyophilized to dryness.

Cryptococcus neoformans capsule regrowth experiments reveal dynamics of enlargement and architecture.

The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded complete characterization. Cryptococcal polysaccharides are known to either remain attached to the cell as capsular polysaccharides (CPSs) or to be shed into the extracellular space as exopolysaccharides (EPSs). While many studies have examined 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 isolated capsular material. We show that sonication or Glucanex enzyme cocktail digestion yields soluble CPS preparations, while use of a French pressure cell press or Glucanex digestion followed by cell disruption removed the capsule and produced cell wall-associated polysaccharide aggregates that we call "capsule ghosts", implying an inherent organization that allows the CPS to exist independent of the cell wall surface. Since sonication and Glucanex digestion were noncytotoxic, it was also possible to observe the cryptococcal cells rebuilding their capsule, revealing the presence of reducing end glycans throughout the capsule. Finally, analysis of dimethyl sulfoxide-extracted and sonicated CPS preparations revealed the conservation of previously identified glucuronoxylomannan motifs only in the sonicated CPS. Together, these observations provide new insights into capsule architecture and synthesis, consistent with a model in which the capsule is assembled from the cell wall outward using smaller polymers, which are then compiled into larger ones.

Hinge influences in murine IgG binding to Cryptococcus neoformans capsule.

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One example is the 3E5 monoclonal murine IgG family, in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes despite their identical variable sequences. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1; these binding patterns have been associated with protective efficacy in murine cryptococcosis. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with an mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in antigen binding could affect antibody biology and engineering.

Inositol Metabolism Regulates Capsule Structure and Virulence in the Human Pathogen Cryptococcus neoformans.

The environmental yeast Cryptococcus neoformans is the most common cause of deadly fungal meningitis in primarily immunocompromised populations. A number of factors contribute to cryptococcal pathogenesis. Among them, inositol utilization has been shown to promote C. neoformans development in nature and invasion of central nervous system during dissemination. The mechanisms of the inositol regulation of fungal virulence remain incompletely understood. In this study, we analyzed inositol-induced capsule growth and the contribution of a unique inositol catabolic pathway in fungal development and virulence. We found that genes involved in the inositol catabolic pathway are highly induced by inositol, and they are also highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. This pathway in C. neoformans contains three genes encoding myo-inositol oxygenases that convert myo-inositol into d-glucuronic acid, a substrate of the pentose phosphate cycle and a component of the polysaccharide capsule. Our mutagenesis analysis demonstrates that inositol catabolism is required for C. neoformans virulence and deletion mutants of myo-inositol oxygenases result in altered capsule growth as well as the polysaccharide structure, including O-acetylation. Our study indicates that the ability to utilize the abundant inositol in the brain may contribute to fungal pathogenesis in this neurotropic fungal pathogen. IMPORTANCE The human pathogen Cryptococcus neoformans is the leading cause of fungal meningitis in primarily immunocompromised populations. Understanding how this environmental organism adapts to the human host to cause deadly infection will guide our development of novel disease control strategies. Our recent studies revealed that inositol utilization by the fungus promotes C. neoformans development in nature and invasion of the central nervous system during infection. The mechanisms of the inositol regulation in fungal virulence remain incompletely understood. In this study, we found that C. neoformans has three genes encoding myo-inositol oxygenase, a key enzyme in the inositol catabolic pathway. Expression of these genes is highly induced by inositol, and they are highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. Our mutagenesis analysis indeed demonstrates that inositol catabolism is required for C. neoformans virulence by altering the growth and structure of polysaccharide capsule, a major virulence factor. Considering the abundance of free inositol and inositol-related metabolites in the brain, our study reveals an important mechanism of host inositol-mediated fungal pathogenesis for this neurotropic fungal pathogen.

Critical Online Service-Learning Pedagogy: Justice in Science Education.

In the year 2020 the world changed dramatically. We went from busy lives spent largely away from home to spending most of our time at home while daily facing deepening national crises. With the violent, needless death of George Floyd, the simmering tensions around race in America boiled over, sending thousands into the streets to protest racial injustices. The world of science education has largely avoided discussing racism in our classes, but we can no longer ignore it. The events of the spring and summer have highlighted our need to integrate conversations and reflections on justice into science education. In this work, we argue that service learning can build this understanding from both theory and experience. Using a critical online service-learning framework, we have developed a service-learning course that incorporates dialogic communication, cross-contextual reflections, and positioning oneself as an ally. This perspective allows science and the community to prioritize relationships and humanity and reflect on our roles as professionals using the online interacting space. This course, taught at the beginning of the pandemic, focuses on critical online service learning for those studying public health. We discuss the challenges we faced moving critical service-learning pedagogy online and the compounding issues brought on by the pandemic itself.

A glycan FRET assay for detection and characterization of catalytic antibodies to the Cryptococcus neoformans capsule.

Classic 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 FRET probes, which we used to discover antibodies with innate glycosidase activity and analyze their enzyme kinetics, including mAb 2H1, the most efficient identified 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. Furthermore, the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labeled by a 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 that the antiphagocytic 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.

Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe.

Chemical biology is an emerging field that enables the study and manipulation of biological systems with probes whose reactivities provide structural insights. The opportunistic fungal pathogen Cryptococcus neoformans possesses a polysaccharide capsule that is a major virulence factor, but is challenging to study. We report here the synthesis of a hydroxylamine-armed fluorescent probe that reacts with reducing glycans and its application to study the architecture of the C. neoformans capsule under a variety of conditions. The probe signal localized intracellularly and at the cell wall-membrane interface, implying the presence of reducing-end glycans at this location where the capsule is attached to the cell body. In contrast, no fluorescence signal was detected in the capsule body. We observed vesicle-like structures containing the reducing-end probe, both intra- and extracellularly, consistent with the importance of vesicles in capsular assembly. Disrupting the capsule with DMSO, ultrasound, or mechanical shear stress resulted in capsule alterations that affected the binding of the probe, as reducing ends were exposed and cell membrane integrity was compromised. Unlike the polysaccharides in the assembled capsule, isolated exopolysaccharides contained reducing ends. The reactivity of the hydroxylamine-armed fluorescent probe suggests a model for capsule assembly whereby reducing ends localize to the cell wall surface, supporting previous findings suggesting that this is an initiation point for capsular assembly. We propose that chemical biology is a promising approach for studying the C. neoformans capsule and its associated polysaccharides to unravel their roles in fungal virulence.

A synthetic glycan array containing Cryptococcus neoformans glucuronoxylomannan capsular polysaccharide fragments allows the mapping of protective epitopes.

A convergent synthetic strategy to Cryptococcus neoformans glucuronoxylomannan (GXM) capsular polysaccharide part structures was developed based on di-, tri-, tetra-, penta- and hexasaccharide thioglycoside building blocks. The approach permitted the synthesis of a library of spacer-containing serotype A and D related GXM oligosaccharide structures, ranging from di- to octadecasaccharides. Ten deprotected GXM compounds (mono- to decasaccharide) were printed onto microarray plates and screened with seventeen mouse monoclonal antibodies (mAbs) to GXM. For the first time a GXM oligosaccharide structure (a serotype A decasaccharide), capable of being recognized by neutralizing forms of these GXM-specific mAbs, has been identified, offering insight into the binding epitopes of a range of protective monoclonal antibodies and furthering our efforts to develop semi-synthetic conjugate vaccine candidates against C. neoformans.

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.

First Draft Genome Sequence of the Pathogenic Fungus Lomentospora prolificans (Formerly Scedosporium prolificans).

Here we describe the sequencing and assembly of the pathogenic fungus Lomentospora prolificans using a combination of short, highly accurate Illumina reads and additional coverage in very long Oxford Nanopore reads. The resulting assembly is highly contiguous, containing a total of 37,627,092 bp with over 98% of the sequence in just 26 scaffolds. Annotation identified 8896 protein-coding genes. Pulsed-field gel analysis suggests that this organism contains at least 7 and possibly 11 chromosomes, the two longest of which have sizes corresponding closely to the sizes of the longest scaffolds, at 6.6 and 5.7 Mb.