Cell wall melanin impedes growth of the Cryptococcus neoformans polysaccharide capsule by sequestering calcium.

Cryptococcus neoformans has emerged as a frontrunner among deadly fungal pathogens and is particularly life-threatening for many HIV-infected individuals with compromised immunity. Multiple virulence factors contribute to the growth and survival of C. neoformans within the human host, the two most prominent of which are the polysaccharide capsule and melanin. As both of these features are associated with the cell wall, we were interested to explore possible cooperative or competitive interactions between these two virulence factors. Whereas capsule thickness had no effect on the rate at which cells became melanized, build-up of the melanin pigment layer resulted in a concomitant loss of polysaccharide material, leaving melanized cells with significantly thinner capsules than their nonmelanized counterparts. When melanin was provided exogenously to cells in a transwell culture system we observed a similar inhibition of capsule growth and maintenance. Our results show that melanin sequesters calcium thereby limiting its availability to form divalent bridges between polysaccharide subunits required for outer capsule assembly. The decreased ability of melanized cells to incorporate exported polysaccharide into the growing capsule correlated with the amount of shed polysaccharide, which could have profound negative impacts on the host immune response.

Loss of Opi3 causes a lipid imbalance that influences the virulence traits of Cryptococcus neoformans but not cryptococcosis.

The basidiomycete fungus Cryptococcus neoformans is a useful model for investigating mechanisms of fungal pathogenesis in mammalian hosts. This pathogen is the causative agent of cryptococcal meningitis in immunocompromised patients and is in the critical priority group of the World Health Organization fungal priority pathogens list. In this study, we employed a mutant lacking the OPI3 gene encoding a methylene-fatty-acyl-phospholipid synthase to characterize the role of phosphatidylcholine (PC) and lipid homeostasis in the virulence of C. neoformans. We first confirmed that OPI3 was required for growth in nutrient limiting conditions, a phenotype that could be rescued with exogenous choline and PC. Additionally, we established that loss of Opi3 and the lack of PC lead to an accumulation of neutral lipids in lipid droplets and alterations in major lipid classes. The growth defect of the opi3Δ mutant was also rescued by sorbitol and polyethylene glycol (PEG), a result consistent with protection of ER function from the stress caused by lipid imbalance. We then examined the impact of Opi3 on virulence and found that the dependence of PC synthesis on Opi3 caused reduced capsule size and this was accompanied by an increase in shed capsule polysaccharide and changes in cell wall composition. Further tests of virulence demonstrated that survival in alveolar macrophages and the ability to cause disease in mice were not impacted by loss of Opi3 despite the choline auxotrophy of the mutant in vitro. Overall, this work establishes the contribution of lipid balance to virulence factor elaboration by C. neoformans and suggests that host choline is sufficient to support proliferation during disease.

Sac1 links phosphoinositide turnover to cryptococcal virulence.

Cryptococcus neoformans is an environmentally acquired fungal pathogen that causes over 140,000 deaths per year. Cryptococcal infection occurs when infectious particles are deposited into the lung, where they encounter host phagocytic cells. C. neoformans may be engulfed by these phagocytes, an important step of infection that leads to outcomes ranging from termination of infection to cryptococcal dissemination. To study this critical process, we screened approximately 4,700 cryptococcal gene deletion mutants for altered uptake, using primary mouse and human phagocytic cells. Among the hits of these two screens, we identified 93 mutants with perturbed uptake in both systems, as well as others with differences in uptake by only one cell type. We further screened the hits for changes in thickness of the capsule, a protective polysaccharide layer around the cell which is an important cryptococcal virulence factor. The combination of our three screens yielded 45 mutants, including one lacking the phosphatidylinositol-4-phosphate phosphatase Sac1. In this work, we implicate Sac1 in both host cell uptake and capsule production. We found that sac1 mutants exhibit lipid trafficking defects, reductions in secretory system function, and changes in capsule size and composition. Many of these changes occur specifically in tissue culture media, highlighting the role of Sac1 phosphatase activity in responding to the stress of host-like conditions. Overall, these findings show how genome-scale screening can identify cellular factors that contribute to our understanding of cryptococcal biology and demonstrate the role of Sac1 in determining fungal virulence.IMPORTANCECryptococcus neoformans is a fungal pathogen with significant impact on global health. Cryptococcal cells inhaled from the environment are deposited into the lungs, where they first contact the human immune system. The interaction between C. neoformans and host cells is critical because this step of infection can determine whether the fungal cells die or proliferate within the human host. Despite the importance of this stage of infection, we have limited knowledge of cryptococcal factors that influence its outcome. In this study, we identify cryptococcal genes that affect uptake by both human and mouse cells. We also identify mutants with altered capsule, a protective coating that surrounds the cells to shield them from the host immune system. Finally, we characterize the role of one gene, SAC1, in these processes. Overall, this study contributes to our understanding of how C. neoformans interacts with and protects itself from host cells.

Growth on Douglas fir media facilitates Cryptococcus virulence factor production and enhances fungal survival against environmental and immune stressors.

The yeasts Cryptococcus neoformans and Cryptococcus gattii are fungal pathogens that can be isolated from the environment, including the surfaces of many plants. Cryptococcus gattii caused an outbreak on Vancouver Island, British Columbia beginning in 1999 that has since spread to the Pacific Northwest of the United States. Coastal Douglas fir (Pseudotsuga menziesii) is an important lumber species and a major component of the ecosystems in this area. Previous research has explored Cryptococcus survival and mating on Douglas fir plants and plant-derived material, but no studies have been done on the production of cryptococcal virulence factors by cells grown on those media. Here, we investigated the effects of growth on Douglas fir-derived media on the production of the polysaccharide capsule and melanin, two of the most important cryptococcal virulence factors. We found that while the capsule was mostly unchanged by growth in Douglas fir media compared to cells grown in defined minimal media, Cryptococcus spp. can use substrates present in Douglas fir to synthesize functional and protective melanin. These results suggest mechanisms by which Cryptococcus species may survive in the environment and emphasize the need to explore how association with Douglas fir trees could affect its epidemiology for human cryptococcosis.

Cryptococcus gattii is a fungal pathogen that can be found in the environment. It is responsible for causing an outbreak in British Columbia, Canada, in the late 90s. In our study, we created media from Douglas fir, a tree commonly found in the affected areas. We examined the production of virulence factors by Cryptococcus cells grown in this media.

Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans.

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.

Quantification of C. neoformans Capsule Diameter.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size varies widely between strains, has the ability to grow rapidly when introduced to stressful or low-nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. Inducing the growth of the C. neoformans capsule is used during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here, we describe one of the standard methods of capsule induction and detail two accepted methods of staining: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we outline how to measure capsule diameter manually and offer a protocol for automated diameter measurement of India ink-stained samples using computational image analysis.

Preparation of Biologically Active Fractions Enriched with Glucuronoxylomannan, the Main Antigen of the Cryptococcal Capsule.

Glucuronoxylomannan (GXM) is the principal capsular component in the Cryptococcus genus. This complex polysaccharide participates in numerous events related to the physiology and pathogenesis of Cryptococcus, which highlights the importance of establishing methods for its isolation and analysis. Conventional methods for GXM isolation have been extensively discussed in the literature. In this chapter, we describe two fast methods for obtaining extracellular fractions enriched with cryptococcal GXM.

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.

Proteome and secretome profiling of zinc availability in Cryptococcus neoformans identifies Wos2 as a subtle influencer of fungal virulence determinants.

BACKGROUND: Fungal infections impact over 25% of the global population. For the opportunistic fungal pathogen, Cryptococcus neoformans, infection leads to cryptococcosis. In the presence of the host, disease is enabled by elaboration of sophisticated virulence determinants, including polysaccharide capsule, melanin, thermotolerance, and extracellular enzymes. Conversely, the host protects itself from fungal invasion by regulating and sequestering transition metals (e.g., iron, zinc, copper) important for microbial growth and survival. RESULTS: Here, we explore the intricate relationship between zinc availability and fungal virulence via mass spectrometry-based quantitative proteomics. We observe a core proteome along with a distinct zinc-regulated protein-level signature demonstrating a shift away from transport and ion binding under zinc-replete conditions towards transcription and metal acquisition under zinc-limited conditions. In addition, we revealed a novel connection among zinc availability, thermotolerance, as well as capsule and melanin production through the detection of a Wos2 ortholog in the secretome under replete conditions. CONCLUSIONS: Overall, we provide new biological insight into cellular remodeling at the protein level of C. neoformans under regulated zinc conditions and uncover a novel connection between zinc homeostasis and fungal virulence determinants.

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.

Cryptococcus gattii evades CD11b-mediated fungal recognition by coating itself with capsular polysaccharides.

Cryptococcus gattii is a capsular pathogenic fungus causing life-threatening cryptococcosis. Although the capsular polysaccharides (CPs) of C. gattii are considered as virulence factors, the physiological significance of CP biosynthesis and of CPs themselves is not fully understood, with many conflicting data reported. First, we demonstrated that CAP gene deletant of C. gattii completely lacked capsule layer and its virulence, and that the strain was susceptible to host-related factors including oxidizing, hypoxic, and hypotrophic conditions in vitro. Extracellular CPs recovered from culture supernatant bound specifically to C. gattii acapsular strains, not to other fungi and immune cells, and rendered them the immune escape effects. In fact, dendritic cells (DCs) did not efficiently uptake the CP-treated acapsular strains, which possessed no visible capsule layer, and a decreased amount of phosphorylated proteins and cytokine levels after the stimulation. DCs recognized C. gattii acapuslar cells via an immune receptor CD11b- and Syk-related pathway; however, CD11b did not bind to CP-treated acapsular cells. These results suggested that CPs support immune evasion by coating antigens on C. gattii and blocking the interaction between CD11b and C. gattii cells. Here, we describe the importance of CPs in pathogenicity and immune evasion mechanisms of C. gattii.

Treatment strategies for cryptococcal infection: challenges, advances and future outlook.

Cryptococcus spp., in particular Cryptococcus neoformans and Cryptococcus gattii, have an enormous impact on human health worldwide. The global burden of cryptococcal meningitis is almost a quarter of a million cases and 181,000 deaths annually, with mortality rates of 100% if infections remain untreated. Despite these alarming statistics, treatment options for cryptococcosis remain limited, with only three major classes of drugs approved for clinical use. Exacerbating the public health burden is the fact that the only new class of antifungal drugs developed in decades, the echinocandins, displays negligible antifungal activity against Cryptococcus spp., and the efficacy of the remaining therapeutics is hampered by host toxicity and pathogen resistance. Here, we describe the current arsenal of antifungal agents and the treatment strategies employed to manage cryptococcal disease. We further elaborate on the recent advances in our understanding of the intrinsic and adaptive resistance mechanisms that are utilized by Cryptococcus spp. to evade therapeutic treatments. Finally, we review potential therapeutic strategies, including combination therapy, the targeting of virulence traits, impairing stress response pathways and modulating host immunity, to effectively treat infections caused by Cryptococcus spp. Overall, understanding of the mechanisms that regulate anti-cryptococcal drug resistance, coupled with advances in genomics technologies and high-throughput screening methodologies, will catalyse innovation and accelerate antifungal drug discovery.

Convergent total synthesis of Cryptococcus neoformans serotype B capsule repeating motif.

Cryptococcus neoformans is an opportunistic fungal pathogen, which is a frequent cause of a life-threatening meningitis in immunocompromised individuals. We report the first total synthesis of the serotype B heptasaccharide repeating motif. The use of di- and trisaccharide building blocks enabled a concise convergent synthesis of the protected 6-O-acetylated repeating motif in three steps. Glycosylations gave total 1,2-trans selectivity, despite the absence of a neighboring participating group. Using our recently disclosed catalyst pre-tuning strategy global deprotection gave the desired 6-O-acetylated heptasaccharide with no saturation by-products, overall in four steps 31% yield. The serotype B glucuronoxylomannan (GXM) glycans accessed in this study will increase the structurally diversity of our GXM microarray, allowing further steps towards the development of semi-synthetic vaccines against cryptococcal infections.

Mapping the intracellular metabolome of yeast biocapsules - Spherical structures of yeast attached to fungal pellets.

Co-culture conditions are beneficial for study due to the advances which arise from symbiotic interactions and which cannot be replicated under pure culture conditions. Here, the focus is on the connection between two fungi - a yeast, Saccharomyces cerevisiae, and a filamentous fungus, Penicillium chrysogenum - in a yeast immobilization system termed' yeast biocapsules', where the yeast and filamentous fungus are strongly attached to one another, forming spherical structures. This co-culture condition hinders filamentous fungal biomass growth, while immobilization of yeast cells continues to increase. The effect of the co-culture condition on endometabolites or intracellular metabolites were tracked during the beginning and end of the yeast biocapsule formation period, and metabolites analyzed by Gas Chromatography-Mass Spectrometry Detector (GC-MSD). Distinct metabolite profiles were found between single culture conditions, involving each organism separately, and with the co-culture condition, where there were differences in 54 endometabolites. Specifically, co-culture condition compounds such as fructose, glycolic acid and glyceric acid were present in higher concentrations at the end of biocapsule formation. These results shed light on the mechanisms and biochemical impact of the interaction between the yeast and filamentous fungus and serve as a basis to apply and further develop yeast biocapsules as a new biotechnological tool with benefits for industry.

Assessing the virulence of Cryptococcus neoformans causing meningitis in HIV infected and uninfected patients in Vietnam.

We previously observed a substantial burden of cryptococcal meningitis in Vietnam atypically arising in individuals who are uninfected with human immunodeficiency virus (HIV). This disease was associated with a single genotype of Cryptococcus neoformans (sequence type [ST]5), which was significantly less common in HIV-infected individuals. Aiming to compare the phenotypic characteristics of ST5 and non-ST5 C. neoformans, we selected 30 representative Vietnamese isolates and compared their in vitro pathogenic potential and in vivo virulence. ST5 and non-ST5 organisms exhibited comparable characteristics with respect to in vitro virulence markers including melanin production, replication at 37°C, and growth in cerebrospinal fluid. However, the ST5 isolates had significantly increased variability in cellular and capsular sizing compared with non-ST5 organisms (P < .001). Counterintuitively, mice infected with ST5 isolates had significantly longer survival with lower fungal burdens at day 7 than non-ST5 isolates. Notably, ST5 isolates induced significantly greater initial inflammatory responses than non-ST5 strains, measured by TNF-α concentrations (P < .001). Despite being generally less virulent in the mouse model, we hypothesize that the significant within strain variation seen in ST5 isolates in the tested phenotypes may represent an evolutionary advantage enabling adaptation to novel niches including apparently immunocompetent human hosts.

Lactoferrin Is Broadly Active against Yeasts and Highly Synergistic with Amphotericin B.

Lactoferrin (LF) is a multifunctional milk protein with antimicrobial activity against a range of pathogens. While numerous studies report that LF is active against fungi, there are considerable differences in the level of antifungal activity and the capacity of LF to interact with other drugs. Here we undertook a comprehensive evaluation of the antifungal spectrum of activity of three defined sources of LF across 22 yeast and 24 mold species and assessed its interactions with six widely used antifungal drugs. LF was broadly and consistently active against all yeast species tested (MICs, 8 to 64 µg/ml), with the extent of activity being strongly affected by iron saturation. LF was synergistic with amphotericin B (AMB) against 19 out of 22 yeast species tested, and synergy was unaffected by iron saturation but was affected by the extent of LF digestion. LF-AMB combination therapy significantly prolonged the survival of Galleria mellonella wax moth larvae infected with Candida albicans or Cryptococcus neoformans and decreased the fungal burden 12- to 25-fold. Evidence that LF directly interacts with the fungal cell surface was seen via scanning electron microscopy, which showed pore formation, hyphal thinning, and major cell collapse in response to LF-AMB synergy. Important virulence mechanisms were disrupted by LF-AMB treatment, which significantly prevented biofilms in C. albicans and C. glabrata, inhibited hyphal development in C. albicans, and reduced cell and capsule size and phenotypic diversity in Cryptococcus Our results demonstrate the potential of LF-AMB as an antifungal treatment that is broadly synergistic against important yeast pathogens, with the synergy being attributed to the presence of one or more LF peptides.

Scanning electron microscopy and machine learning reveal heterogeneity in capsular morphotypes of the human pathogen Cryptococcus spp.

Phenotypic heterogeneity is an important trait for the development and survival of many microorganisms including the yeast Cryptococcus spp., a deadly pathogen spread worldwide. Here, we have applied scanning electron microscopy (SEM) to define four Cryptococcus spp. capsule morphotypes, namely Regular, Spiky, Bald, and Phantom. These morphotypes were persistently observed in varying proportions among yeast isolates. To assess the distribution of such morphotypes we implemented an automated pipeline capable of (1) identifying potentially cell-associated objects in the SEM-derived images; (2) computing object-level features; and (3) classifying these objects into their corresponding classes. The machine learning approach used a Random Forest (RF) classifier whose overall accuracy reached 85% on the test dataset, with per-class specificity above 90%, and sensitivity between 66 and 94%. Additionally, the RF model indicates that structural and texture features, e.g., object area, eccentricity, and contrast, are most relevant for classification. The RF results agree with the observed variation in these features, consistently also with visual inspection of SEM images. Finally, our work introduces morphological variants of Cryptococcus spp. capsule. These can be promptly identified and characterized using computational models so that future work may unveil morphological associations with yeast virulence.

Impact of iron chelators on growth and expression of iron-related genes of Cryptococcus species.

INTRODUCTION: Iron chelator has previously demonstrated fungicidal effects. This study aimed to investigate the antifungal activity of the iron chelators deferoxamine (DFO) and deferasirox (DSX) against Cryptococcus. MATERIALS AND METHODS: Cryptococcus neoformans and Cryptococcus gattii were used to determine the minimal inhibitory concentrations (MICs) of DFO and DSX, and the fractional inhibitory concentration index (FICI) of DFO and DSX when combined with amphotericin B (AMB). Expression of cryptococcal CFT1, CFT2, and CIR1 genes was determined using real-time polymerase chain reaction (PCR). RESULTS: Neither DFO nor DSX alone showed antifungal activity against Cryptococcus strains. When combined with AMB, the MICs of DFO and DSX decreased from>200µg/mL to 6.25 or 12.5µg/mL. The MIC of AMB decreased one-fold dilution in most strains when combined with iron chelators. The FICI of DFO+AMB and DSX+AMB was 0.5 and 1, respectively. C. neoformans showed significant growth retardation when incubated with a combination of sub-MIC concentrations of AMB and DFO; whereas, C. gattii demonstrated lesser growth retardation in DFO+AMB. No cryptococcal growth retardation was observed when DSX was combined with AMB. When C. neoformans was grown in DFO, the CFT1, CFT2, and CIR1 proteins were expressed 1.7, 2.0, and 0.9 times, respectively. When C. neoformans was grown in DSX, the CFT1, CFT2, and CIR1 genes were expressed 0.5, 0.6, and 0.3 times, respectively. CONCLUSION: Synergistic antifungal activity of combination DFO and AMB was observed in Cryptococcus. Relatively increased CFT1 and CFT2 expression may be associated with the effect of DFO that inhibits the growth of fungi.

Rheological properties of cryptococcal polysaccharide change with fiber size, antibody binding and temperature.

Aim:Cryptococcus neoformans is the major agent of cryptococcosis. The main virulence factor is the polysaccharide (PS) capsule. Changes in cryptococcal PS properties have been poorly elucidated. Materials & methods: We analyzed the mechanical properties of secreted PS and intact capsules, using dynamic light scattering and optical tweezers. Results: Storage and loss moduli showed that secreted PS behaves as a viscoelastic liquid, while capsular PS behaves as a viscoelastic solid. The secreted PS remains as a viscoelastic fluid at different temperatures with thermal hysteresis after 85°C. Antibody binding altered the viscoelastic behavior of both secreted and capsular PS. Conclusion: Deciphering the mechanical aspects of these structures could reveal features that may have consequences in novel therapies against cryptococcosis.

Reduced phagocytosis and killing of Cryptococcus neoformans biofilm-derived cells by J774.16 macrophages is associated with fungal capsular production and surface modification.

Cryptococcus neoformans is an opportunistic encapsulated pathogen that causes life-threatening meningoencephalitis in individuals with immunosuppression. We compared the interactions of C. neoformans planktonic and biofilm-derived cells with J774.16 macrophage-like cells. Planktonic cells are more phagocytized and killed by J774.16 cells than biofilm-derived fungal cells. Biofilm-derived cryptococci possess larger capsule size and release significantly more capsular polysaccharide than planktonic cells in culture. Biofilm-derived fungi exhibited upregulation of genes involved in capsular production. Capsular-specific monoclonal antibody 18B7 demonstrated differential binding to the surface of planktonic and biofilm-derived cryptococci providing a plausible strategy for fungal evasion of macrophages and persistence. Future studies are necessary to elucidate how C. neoformans biofilm-derived cells regulate their virulence factors when interacting with cells of the immune system.