Unraveling the biology of a fungal meningitis pathogen using chemical genetics.

The fungal meningitis pathogen Cryptococcus neoformans is a central driver of mortality in HIV/AIDS. We report a genome-scale chemical genetic data map for this pathogen that quantifies the impact of 439 small-molecule challenges on 1,448 gene knockouts. We identified chemical phenotypes for 83% of mutants screened and at least one genetic response for each compound. C. neoformans chemical-genetic responses are largely distinct from orthologous published profiles of Saccharomyces cerevisiae, demonstrating the importance of pathogen-centered studies. We used the chemical-genetic matrix to predict novel pathogenicity genes, infer compound mode of action, and to develop an algorithm, O2M, that predicts antifungal synergies. These predictions were experimentally validated, thereby identifying virulence genes, a molecule that triggers G2/M arrest and inhibits the Cdc25 phosphatase, and many compounds that synergize with the antifungal drug fluconazole. Our work establishes a chemical-genetic foundation for approaching an infection responsible for greater than one-third of AIDS-related deaths.

Secreted fungal virulence effector triggers allergic inflammation via TLR4.

Invasive fungal pathogens are major causes of human mortality and morbidity1,2. Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans, the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages3-8. Here, we identify CPL1 as an effector protein secreted by C. neoformans that drives alternative activation (also known as M2 polarization) of macrophages to enable pulmonary infection in mice. We observed that CPL1-enhanced macrophage polarization requires Toll-like receptor 4, which is best known as a receptor for bacterial endotoxin but is also a poorly understood mediator of allergen-induced type 2 responses9-12. We show that this effect is caused by CPL1 itself and not by contaminating lipopolysaccharide. CPL1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signalling for its effect on infectivity. Notably, C. neoformans associates selectively with polarized interstitial macrophages during infection, suggesting a mechanism by which C. neoformans generates its own intracellular replication niche within the host. This work identifies a circuit whereby a secreted effector protein produced by a human fungal pathogen reprograms innate immunity, revealing an unexpected role for Toll-like receptor 4 in promoting the pathogenesis of infectious disease.

Glyphosate inhibits melanization and increases susceptibility to infection in insects.

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin's broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate-the most widely used herbicide globally-inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate's mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance. Overall, these findings suggest that glyphosate's environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.

Pleiotropy and epistasis within and between signaling pathways defines the genetic architecture of fungal virulence.

Cryptococcal disease is estimated to affect nearly a quarter of a million people annually. Environmental isolates of Cryptococcus deneoformans, which make up 15 to 30% of clinical infections in temperate climates such as Europe, vary in their pathogenicity, ranging from benign to hyper-virulent. Key traits that contribute to virulence, such as the production of the pigment melanin, an extracellular polysaccharide capsule, and the ability to grow at human body temperature have been identified, yet little is known about the genetic basis of variation in such traits. Here we investigate the genetic basis of melanization, capsule size, thermal tolerance, oxidative stress resistance, and antifungal drug sensitivity using quantitative trait locus (QTL) mapping in progeny derived from a cross between two divergent C. deneoformans strains. Using a "function-valued" QTL analysis framework that exploits both time-series information and growth differences across multiple environments, we identified QTL for each of these virulence traits and drug susceptibility. For three QTL we identified the underlying genes and nucleotide differences that govern variation in virulence traits. One of these genes, RIC8, which encodes a regulator of cAMP-PKA signaling, contributes to variation in four virulence traits: melanization, capsule size, thermal tolerance, and resistance to oxidative stress. Two major effect QTL for amphotericin B resistance map to the genes SSK1 and SSK2, which encode key components of the HOG pathway, a fungal-specific signal transduction network that orchestrates cellular responses to osmotic and other stresses. We also discovered complex epistatic interactions within and between genes in the HOG and cAMP-PKA pathways that regulate antifungal drug resistance and resistance to oxidative stress. Our findings advance the understanding of virulence traits among diverse lineages of Cryptococcus, and highlight the role of genetic variation in key stress-responsive signaling pathways as a major contributor to phenotypic variation.

Small-molecule inhibitors for the Prp8 intein as antifungal agents.

Self-splicing proteins, called inteins, are present in many human pathogens, including the emerging fungal threats Cryptococcus neoformans (Cne) and Cryptococcus gattii (Cga), the causative agents of cryptococcosis. Inhibition of protein splicing in Cryptococcus sp. interferes with activity of the only intein-containing protein, Prp8, an essential intron splicing factor. Here, we screened a small-molecule library to find addititonal, potent inhibitors of the Cne Prp8 intein using a split-GFP splicing assay. This revealed the compound 6G-318S, with IC50 values in the low micromolar range in the split-GFP assay and in a complementary split-luciferase system. A fluoride derivative of the compound 6G-318S displayed improved cytotoxicity in human lung carcinoma cells, although there was a slight reduction in the inhibition of splicing. 6G-318S and its derivative inhibited splicing of the Cne Prp8 intein in vivo in Escherichia coli and in C. neoformans Moreover, the compounds repressed growth of WT C. neoformans and C. gattii In contrast, the inhibitors were less potent at inhibiting growth of the inteinless Candida albicans Drug resistance was observed when the Prp8 intein was overexpressed in C. neoformans, indicating specificity of this molecule toward the target. No off-target activity was observed, such as inhibition of serine/cysteine proteases. The inhibitors bound covalently to the Prp8 intein and binding was reduced when the active-site residue Cys1 was mutated. 6G-318S showed a synergistic effect with amphotericin B and additive to indifferent effects with a few other clinically used antimycotics. Overall, the identification of these small-molecule intein-splicing inhibitors opens up prospects for a new class of antifungals.

The granuloma in cryptococcal disease.

Although we have recognized cryptococcosis as a disease entity for well over 100 years, there are many details about its pathogenesis which remain unknown. A major barrier to better understanding is the very broad range of clinical and pathological forms cryptococcal infections can take. One such form has been historically called the cryptococcal granuloma, or the cryptococcoma. These words have been used to describe essentially any mass lesion associated with infection, due to their presumed similarity to the quintessential granuloma, the tubercle in tuberculosis. Although clear distinctions between tuberculosis and cryptococcal disease have been discovered, cellular and molecular studies still confirm some important parallels between these 2 diseases and what we now call granulomatous inflammation. In this review, we shall sketch out some of the history behind the term "granuloma" as it pertains to cryptococcal disease, explore our current understanding of the biology of granuloma formation, and try to place that understanding in the context of the myriad pathological presentations of this infection. Finally, we shall summarize the role of the granuloma in cryptococcal latency and present opportunities for future investigations.

Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans.

The fungus Cryptococcus neoformans is a leading cause of mortality and morbidity among HIV-infected individuals. We utilized the completed genome sequence and optimized methods for homologous DNA replacement using high-velocity particle bombardment to engineer 1201 gene knockout mutants. We screened this resource in vivo for proliferation in murine lung tissue and in vitro for three well-recognized virulence attributes-polysaccharide capsule formation, melanization, and growth at body temperature. We identified dozens of previously uncharacterized genes that affect these known attributes as well as 40 infectivity mutants without obvious defects in these traits. The latter mutants affect predicted regulatory factors, secreted proteins, and immune-related factors, and represent powerful tools for elucidating novel virulence mechanisms. In particular, we describe a GATA family transcription factor that inhibits phagocytosis by murine macrophages independently of the capsule, indicating a previously unknown mechanism of innate immune modulation.

Centromere scission drives chromosome shuffling and reproductive isolation.

A fundamental characteristic of eukaryotic organisms is the generation of genetic variation via sexual reproduction. Conversely, significant large-scale genome structure variations could hamper sexual reproduction, causing reproductive isolation and promoting speciation. The underlying processes behind large-scale genome rearrangements are not well understood and include chromosome translocations involving centromeres. Recent genomic studies in the Cryptococcus species complex revealed that chromosome translocations generated via centromere recombination have reshaped the genomes of different species. In this study, multiple DNA double-strand breaks (DSBs) were generated via the CRISPR/Cas9 system at centromere-specific retrotransposons in the human fungal pathogen Cryptococcus neoformans The resulting DSBs were repaired in a complex manner, leading to the formation of multiple interchromosomal rearrangements and new telomeres, similar to chromothripsis-like events. The newly generated strains harboring chromosome translocations exhibited normal vegetative growth but failed to undergo successful sexual reproduction with the parental wild-type strain. One of these strains failed to produce any spores, while another produced ∼3% viable progeny. The germinated progeny exhibited aneuploidy for multiple chromosomes and showed improved fertility with both parents. All chromosome translocation events were accompanied without any detectable change in gene sequences and thus suggest that chromosomal translocations alone may play an underappreciated role in the onset of reproductive isolation and speciation.

Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro.

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 (rap/FK506) and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37° compared to 30° in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNA interference (RNAi) pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30° to the level observed at 37°. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host.

Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

Role of the anillin-like protein in growth of Cryptococcus neoformans at human host temperature.

Cryptococcus neoformans, a basidiomycete yeast, causes lethal meningitis in immunocompromised individuals. The ability of C. neoformans to proliferate at 37°C is essential for virulence. We identified anillin-like protein, CnBud4, as essential for proliferation of C. neoformans at 37°C and for virulence in a heterologous host Galleria mellonella at 25°C. C. neoformans cells lacking CnBud4 were inviable at 25°C in the absence of active calcineurin and were hypersensitive to membrane stress and an anti-fungal agent fluconazole, phenotypes previously described for C. neoformans mutants lacking septins. CnBud4 localized to the mother-bud neck during cytokinesis in a septin-dependent manner. In the absence of CnBud4, septin complex failed to transition from a collar-like single ring to the double ring during cytokinesis. In an ascomycete yeast, Saccharomyces cerevisiae, the anillin-like homologue ScBud4 participates in the organization of the septin ring at the mother-bud neck and plays an important role in specifying location for new bud emergence, known as axial budding pattern. In contrast to their role in S. cerevisiae, neither septins nor CnBud4 were needed to direct the position of the new bud in C. neoformans, suggesting that this function is not conserved in basidiomycetous yeasts. Our data suggest that the requirement of CnBud4 for growth at 37°C and pathogenicity in C. neoformans is based on its conserved role in septin complex organization.

VCAM1/VLA4 interaction mediates Ly6Clow monocyte recruitment to the brain in a TNFR signaling dependent manner during fungal infection.

Monocytes exist in two major populations, termed Ly6Chi and Ly6Clow monocytes. Compared to Ly6Chi monocytes, less is known about Ly6Clow monocyte recruitment and mechanisms involved in the recruitment of this subset. Furthermore, the role of Ly6Clow monocytes during infections is largely unknown. Here, using intravital microscopy, we demonstrate that Ly6Clow monocytes are predominantly recruited to the brain vasculature following intravenous infection with Cryptococcus neoformans, a fungal pathogen causing meningoencephalitis. The recruitment depends primarily on the interaction of VCAM1 expressed on the brain endothelium with VLA4 expressed on Ly6Clow monocytes. Furthermore, TNFR signaling is essential for the recruitment through enhancing VLA4 expression on Ly6Clow monocytes. Interestingly, the recruited Ly6Clow monocytes internalized C. neoformans and carried the organism while crawling on and adhering to the luminal wall of brain vasculature and migrating to the brain parenchyma. Our study reveals a substantial recruitment of Ly6Clow monocytes to the brain and highlights important properties of this subset during infection.

Viral infection triggers interferon-induced expulsion of live Cryptococcus neoformans by macrophages.

Cryptococcus neoformans is an opportunistic human pathogen, which causes serious disease in immunocompromised hosts. Infection with this pathogen is particularly relevant in HIV+ patients, where it leads to around 200,000 deaths per annum. A key feature of cryptococcal pathogenesis is the ability of the fungus to survive and replicate within the phagosome of macrophages, as well as its ability to be expelled from host cells via a novel non-lytic mechanism known as vomocytosis. Here we show that cryptococcal vomocytosis from macrophages is strongly enhanced by viral coinfection, without altering phagocytosis or intracellular proliferation of the fungus. This effect occurs with distinct, unrelated human viral pathogens and is recapitulated when macrophages are stimulated with the anti-viral cytokines interferon alpha or beta (IFNα or IFNß). Importantly, the effect is abrogated when type-I interferon signalling is blocked, thus underscoring the importance of type-I interferons in this phenomenon. Lastly, our data help resolve previous, contradictory animal studies on the impact of type I interferons on cryptococcal pathogenesis and suggest that secondary viral stimuli may alter patterns of cryptococcal dissemination in the host.

Caenorhabditis elegans DAF-16 regulates lifespan and immune responses to Cryptococcus neoformans and Cryptococcus gattii infections.

BACKGROUND: Cryptococcosis is a life-threatening infection is primarily caused by two sibling species Cryptococcus neoformans and Cryptococcus gattii. Several virulence-related factors of these cryptococci have been widely investigated in Caenorhabditis elegans, representing a facile in vivo model of host-pathogen interaction. While recent studies elucidated cryptococcal virulence factors, intrinsic host factors that affect susceptibility to infections by cryptococci remain unclear and poorly investigated. RESULTS: Here, we showed that defects in C. elegans insulin/insulin-like growth factor-1 (IGF-1) signaling (IIS) pathway influenced animal lifespan and mechanisms of host resistance in cryptococcal infections, which required the activation of aging regulator DAF-16/Forkhead box O transcription factor. Moreover, accumulation of lipofuscin, DAF-16 nuclear localization, and expression of superoxide dismutase (SOD-3) were elevated in C. elegans due to host defenses during cryptococcal infections. CONCLUSION: The present study demonstrated the relationship between longevity and immunity, which may provide a possibility for novel therapeutic intervention to improve host resistance against cryptococcal infections.

A lytic polysaccharide monooxygenase-like protein functions in fungal copper import and meningitis.

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu+ import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1, that is strongly induced during Cu limitation and which encodes a protein related to lytic polysaccharide monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient, and Bim1 function in Cu accumulation depends on Cu2+ coordination and cell-surface association via a glycophosphatidyl inositol anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis.

Quantitative analysis reveals internalisation of Cryptococcus neoformans by brain endothelial cells in vivo.

Migration of Cryptococcus neoformans from the blood to the brain parenchyma is crucial to cause fatal meningoencephalitis. Although mechanisms involved in brain migration of C. neoformans have been widely studied in vitro, less is known about how the fungus crosses the blood-brain barrier (BBB) in vivo. This is in part because of the lack of an approach to quantitatively analyse the dynamics of fungal transmigration into the brain across the BBB in vivo. In this study, we report a novel approach to quantitatively analyse the interactions between C. neoformans and brain endothelial cells in a mouse model using flow cytometry. Using this system, we show that C. neoformans was internalised by brain endothelial cells in vivo and that mice infected with acapsular or heat-killed C. neoformans yeast cells displayed a lower frequency of brain endothelial cells containing the yeast cell compared to mice infected with wild-type or viable yeast cells, respectively. We further demonstrate that brain endothelial cells were invaded by serotype A strain (H99 strain) at a higher rate compared to serotype D strain (52D strain). Our experiments established that internalisation of C. neoformans by brain endothelial cells occurred in vivo and offered a powerful approach to quantitatively analyse fungal migration into the brain.

Unisexual reproduction promotes competition for mating partners in the global human fungal pathogen Cryptococcus deneoformans.

Courtship is pivotal for successful mating. However, courtship is challenging for the Cryptococcus neoformans species complex, comprised of opportunistic fungal pathogens, as the majority of isolates are α mating type. In the absence of mating partners of the opposite mating type, C. deneoformans can undergo unisexual reproduction, during which a yeast-to-hyphal morphological transition occurs. Hyphal growth during unisexual reproduction is a quantitative trait, which reflects a strain's ability to undergo unisexual reproduction. In this study, we determined whether unisexual reproduction confers an ecological benefit by promoting foraging for mating partners. Through competitive mating assays using strains with different abilities to produce hyphae, we showed that unisexual reproduction potential did not enhance competition for mating partners of the same mating type, but when cells of the opposite mating type were present, cells with enhanced hyphal growth were more competitive for mating partners of either the same or opposite mating type. Enhanced mating competition was also observed in a strain with increased hyphal production that lacks the mating repressor gene GPA3, which contributes to the pheromone response. Hyphal growth in unisexual strains also enables contact between adjacent colonies and enhances mating efficiency during mating confrontation assays. The pheromone response pathway activation positively correlated with unisexual reproduction hyphal growth during bisexual mating and exogenous pheromone promoted bisexual cell fusion. Despite the benefit in competing for mating partners, unisexual reproduction conferred a fitness cost. Taken together, these findings suggest C. deneoformans employs hyphal growth to facilitate contact between colonies at long distances and utilizes pheromone sensing to enhance mating competition.

Solid-state NMR spectroscopy identifies three classes of lipids in Cryptococcus neoformans melanized cell walls and whole fungal cells.

A primary virulence-associated trait of the opportunistic fungal pathogen Cryptococcus neoformans is the production of melanin pigments that are deposited into the cell wall and interfere with the host immune response. Previously, our solid-state NMR studies of isolated melanized cell walls (melanin "ghosts") revealed that the pigments are strongly associated with lipids, but their identities, origins, and potential roles were undetermined. Herein, we exploited spectral editing techniques to identify and quantify the lipid molecules associated with pigments in melanin ghosts. The lipid profiles were remarkably similar in whole C. neoformans cells, grown under either melanizing or nonmelanizing conditions; triglycerides (TGs), sterol esters (SEs), and polyisoprenoids (PPs) were the major constituents. Although no quantitative differences were found between melanized and nonmelanized cells, melanin ghosts were relatively enriched in SEs and PPs. In contrast to lipid structures reported during early stages of fungal growth in nutrient-rich media, variants found herein could be linked to nutrient stress, cell aging, and subsequent production of substances that promote chronic fungal infections. The fact that TGs and SEs are the typical cargo of lipid droplets suggests that these organelles could be connected to C. neoformans melanin synthesis. Moreover, the discovery of PPs is intriguing because dolichol is a well-established constituent of human neuromelanin. The presence of these lipid species even in nonmelanized cells suggests that they could be produced constitutively under stress conditions in anticipation of melanin synthesis. These findings demonstrate that C. neoformans lipids are more varied compositionally and functionally than previously recognized.

Melanin deposition in two Cryptococcus species depends on cell-wall composition and flexibility.

Cryptococcus neoformans and Cryptococcus gattii are two species complexes in the large fungal genus Cryptococcus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In C. neoformans, the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall. Consequently, melanization is modulated by changes in cell-wall composition or ultrastructure. However, whether similar factors influence melanization in C. gattii is unknown. Herein, we used transmission EM, biochemical assays, and solid-state NMR spectroscopy of representative isolates and "leaky melanin" mutant strains from each species complex to examine the compositional and structural factors governing cell-wall pigment deposition in C. neoformans and C. gattii. The principal findings were the following. 1) C. gattii R265 had an exceptionally high chitosan content compared with C. neoformans H99; a rich chitosan composition promoted homogeneous melanin distribution throughout the cell wall but did not increase the propensity of pigment deposition. 2) Strains from both species manifesting the leaky melanin phenotype had reduced chitosan content, which was compensated for by the production of lipids and other nonpolysaccharide constituents that depended on the species or mutation. 3) Changes in the relative rigidity of cell-wall chitin were associated with aberrant pigment retention, implicating cell-wall flexibility as an independent variable in cryptococcal melanin assembly. Overall, our results indicate that cell-wall composition and molecular architecture are critical factors for the anchoring and arrangement of melanin pigments in both C. neoformans and C. gattii species complexes.

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.